JPH06113833A - A new retrovirus that causes AIDS - Google Patents

Abstract

PURPOSE: To obtain a virus of the class having the ability to cause lymphadenopathy developed into human acquired immunodeficiency syndrome(AIDS).

CONSTITUTION: This invention relates to an antigen, infectious with a human T4 lymphocyte and recognizable with an antibody induced in a human by an HIV-2 retrovirus having essential morphological and immunological characteristics of a retrovirus deposited in CNCM as accession number I-502, I-532, I-642 or I-643. The antibody is induced by the antibody obtained from the virus.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

The present invention relates to a novel class of viruses with the ability to develop lymph node disorders that develop into human acquired immunodeficiency syndrome (AIDS). The present invention also relates to antigens that can be recognized by antibodies elicited in humans by this novel class of viruses. The present invention also relates to antibodies elicited by the antigens obtained from these viruses.

The invention further relates to the genomic RNA of said virus.
To a cloned DNA sequence having sequence similarity or complementarity with. The present invention also relates to methods for preparing these cloned DNA sequences.

The invention further relates to a polypeptide comprising an amino acid sequence encoded by said cloned DNA sequence.

Furthermore, the present invention relates to the use of the antigens of the invention for the in vitro diagnosis of several forms of AIDS latent in humans, and also to immunogens against retroviruses using some of these antigens. And a vaccination composition. The invention also relates to the use of said antibody for the same purpose as said antigen and to the production of the active ingredient of the drug against various forms of human AIDS using some of these antibodies.

Finally, the present invention includes a use invention in which a cloned DNA sequence and a polypeptide obtained from the sequence are used as a probe for a diagnostic kit.

LA is recognized as the cause of AIDS
The isolation and characterization of the first retrovirus known as V was previously published in a paper by F. BARRE-SINOUSSI et al. In 1983 (Science, vol. 220, No. 45-49, 2
0, p.868-871). Also, the use of certain extracts of said LAV virus and in particular certain proteins for diagnosing the presence of antibodies against the AIDS virus is described in detail in EP-A-138667. Then LA
Another strain similar to V and several mutant strains were isolated. Examples of these are designated as HTLV-III and ARV.

In accordance with the new nomenclature published in Nature in May 1986, the retrovirus capable of inducing the above-mentioned lymph node disorders and AIDS in humans has been designated as a general name HIV. This is an abbreviation for "Human Immunodeficiency Virus". The subgroup of retroviruses formed by LAV and its variants was originally designated "LAV type I" or "LAV-I". In the present text, this subgroup will be referred to as HIV-1 hereinafter, but among the retrovirus strains belonging to the HIV-1 virus class (in particular LAAV, IDAV-4 and IDAV-2), the above-mentioned European Patent Application No. 138667 is referred to. LAV when referring to the listed strains
It should be understood that the term This strain was used as LAV _BRU in the comparative experiments described below.
Coll at the Pasteur Institute in Paris, France on July 15, 2016
ection Nationale des Cultures de Micro-Organismes (C
NCM) with deposit number I-232.

The novel retroviruses that form the subject of the present invention, and related viral strains which have hitherto been referred to as "LAV type II" or "LAV-II" and which are able to grow in human lymphocytes like the retroviruses of the present invention are hereinafter referred to as "HIV. -2 ".
However, it is to be understood that certain HIV-2 isolates described below are designated with a three letter designation indicating the patient who provided the isolate.

[0009] The "HIV-2" group contains human T4 in vitro.
It can be defined as a group of viruses that have an affinity for lymphocytes and have a cytopathic effect on these lymphocytes when they grow in human lymphocytes, causing systemic and persistent polydactyly or one form of AIDS. It was demonstrated that the HIV-2 retrovirus differs from the HIV-1 type virus under the conditions described below. Also, both HIV-2 retroviruses and HIV-1 type viruses differ from other known human retroviruses (HTLV-I and HTLV-II).

Although there is fairly widespread genetic dispersal in the virus, to date various HIV-1 strains isolated from American, European, Haiti and African patients share a common antigenic site. This site is the major protein, core protein p25, envelope glycoprotein gp110.
And is held by the transmembrane protein gp41-43. Thus, it is possible, for example, to use the prototypical LAV strain as an antigen strain for the detection of antibodies against all HIV-1 class viruses in all carriers, regardless of their origin. Therefore, this strain is used especially for detecting anti-HIV-1 antibody in blood donors and patients by using immunofluorescence method known as ELISA method, Western blot (immunoimprinting) method, RIPA (immunoprecipitation assay) method and the like. Widely used.

However, the HI of patients from West Africa
Serological studies performed on V-1 lysates showed that these lysates showed clinically and immunologically signs of AIDS, but serologic reactions were negative or very weakly positive. It was

HIV-2 was first isolated from cultured lymphocytes in one of these patients. HI isolated from this source
The structure of V-2 observed by electron microscopy and the protein profile on SDS gel electrophoresis were similar to HIV-1. However, overall, the novel retrovirus HIV-2 is poorly associated with HIV-1 both in terms of protein antigen homology and genetic material homology.

This new retrovirus, or a retrovirus having antigenic and immunological properties equivalent thereto, is used for diagnosing the infection of a virus or a virus mutant which induces the AIDS morphology, which was first detected from African residents or people who have been in Africa. Configure an antigen source for

The virus was typically isolated from blood taken in the presence of heparin from a 28-year-old heterosexual patient with no experience of transfusion and drug addiction. The patient has had considerable diarrhea since 1983, with occasional fever and extreme weight loss (17 kg). Then Candida and Serratia
Esophageal candidiasis (can) which is typical of AIDS and is infected with Seratia
didiasis).

This patient also exhibits anemia, cutaneous anergy and lymphopenia with a T4 / T8 lymphocyte ratio.
At 0.15 T4 lymphocyte levels are less than 100 per mm ³ of serum. Cultured lymphocytes did not respond to phytohemagglutinin and concanavalin A stimulation. This patient is also S.en
recurrent bacteremia due to teriditis , cryptosporidioses,
He suffered from infectious disease caused by war Isospora belli and cerebral toxoplasmosis.

These concomitant symptoms are the type of “AIDS-related syndrome” or ARC (AIDS-R) caused by the HIV-1 virus.
It is a sign of elated Complex). These various findings are also based on the Center of Disease Control (CD
It also agreed with the criteria adopted in C).

In order to culture lymphocytes from patients and isolate retroviruses, the method for isolating HIV-1 described in the article of BARRE-SINOUSSI and European Patent Application No. 84 / 401.834-0.138.667 was used. To do. These methods will be briefly described below. Lymphocytes stimulated with phytohemagglutinin (PHA) for 3 days were treated with RPMI1640 medium supplemented with 10% fetal bovine serum, 10 ^-5 M β-mercaptoethanol, interleukin-2 and anti- (human interferon α) serum. Culture at.

Virus production was followed by reverse transcriptase activity. Peak virus activity in culture supernatant from 14 days
It occurred in 22 days and decreased thereafter. Cell culture decay and mortality were followed. When observed with an electron microscope, the section of lymphocytes infected with HIV-2 has virions that have reached maturation, and virus particles are budding on the surface of infected cells, as in the case of HIV-1. The cell line used to produce cultures of these isolated viruses may optionally be a HUT, CEM or MOLT type cell line or any established lymphocyte cell line carrying the T4 receptor.

The virus was then grown in cultures of donor lymphocytes and then in a continuous cell line of leukemic origin such as HUT78. Characterization has revealed that this antigen and nucleic acid differs substantially from that of HIV-1.
The virus was purified by the method described in the conventional literature as described above. The first isolate of this virus was deposited with the CNCM under accession number I-502 on December 19, 1985 under the Budapest Treaty and subsequently named LAV-II MIR. The second isolate was also deposited with the CNCM under the Budapest Treaty on February 21, 1986, with accession number I-532, and was named LAV-II ROD.
These isolates are often referred to simply as MIR or ROD.

In general, the present invention relates to any equivalent virus containing structural proteins having the same immunological properties as the HIV-2 virus deposited at the CNCM as I-502 or I-532 (for example, December 19, 1986). Accession number I- under the Budapest Treaty
HIV-2-IRMO and HIV-2 deposited at 642 and I-643 respectively
-EHO) and any variants thereof. The definition of the equivalence criteria will be described later.

The present invention also relates to HIV-2 in a permanent cell line derived from T4 lymphocytes or lymphocytes carrying the T4 phenotype.
It relates to a method for producing a virus or a mutant strain thereof. In this method, these cell lines pre-infected with the HIV-2 virus are cultured and the amount of virus released in the medium is recovered, especially when the level of reverse transcriptase activity reaches a certain threshold.

A preferred permanent cell line for culturing HIV-2 is, for example, the HUT78 cell type. Infected with HIV-2
The HUT78 line was deposited at the CNCM on February 6, 1986 with accession number I-519. The culture is performed as follows, for example.

Infected HUT78 cells (10 ⁶ / ml) are co-cultured with infected normal human lymphocytes (10 ⁶ / ml). The medium is RPMI 1640 containing 10% fetal bovine serum. Observe the cytopathic effect in HUT78 cells after 15-21 days. One week after this observation, the reverse transcriptase in the medium supernatant is quantified, and virus recovery from the supernatant is started.

Another cell line suitable for culture is the cell line known by the designation CEM.

Infection of CEM cells and culturing of infected CEM cells is carried out as follows.

T4 lymphocytes pre-infected with HIV-2 virus and non-infected CEM cells are co-cultured for a period required for CEM infection. Next, the culture conditions are maintained in an appropriate medium as described below, for example, and when the reverse transcriptase activity of the infected cells reaches a sufficient level, the produced virus is recovered from the medium.

In particular, human T4 lymphocytes previously infected with a HIV-2 strain derived from a patient referred to as ROD in the present text for 5 days and CEM are co-cultured under the conditions described below.

Infected T4 lymphocytes pre-activated with phytohemagglutinin were shown to have a reverse transcriptase activity of 5000 cpm per 10 ⁶ normal T lymphocytes 3 days after initiation of infection. The culture was continued until the measured value of reverse transcriptase activity in the supernatant reached 100,000 cpm. These T4 lymphocytes were then contacted with CEM cells (3 × 10 ⁶ infected normal T lymphocytes) and reincubated in the following medium. Medium: 2.92 mg / ml L-glutamine, 10% complement-free fetal bovine serum, 2 μg / ml Polybrene, 0.05% anti-interferon alpha serum, 100,000 μg / ml penicillin and 10 μg / ml streptomycin And RPMI containing 10,000 μg / ml neomycin
1640.

The medium is changed twice a week.

The measured values of reverse transcriptase activity measured in the supernatant are shown below.

0 days 1,000 (background) 15 days 20,000 21 days 200,000 35 days 1,000,000 HIV-2 virus-infected CEM cultures were submitted to the CNCM of Pasteur Institute on March 24, 1986 under accession number I-537. Deposited.

Some properties of the antigens and nucleic acids involved in the structure of HIV-2 were revealed by experiments carried out under the following conditions. In many cases these properties are more pronounced by comparison with the same properties in other types of retroviruses, especially HIV-1 and SIV.

Next, the attached drawings will be described.

FIGS. 1A, B and C show HIV-1 and HIV-2.
Cross-precipitation experiments of HIV-1 virus extract with sera of patients infected with HIV and STLV-III infected rhesus monkeys, respectively.

FIGS. 2A and 2B show the comparison results of the electrophoretic mobilities of the respective proteins of HIV-1, HIV-2 and STLV-III in SDS polyacrylamide gel.

FIG. 3 shows the results of cross-hybridization between probes containing the HIV-1, HIV-2 and STLV-III genomic sequences and various subgenomic sequences of the HIV-1 virus.

FIG. 4 is derived from HIV-2 ROD RNA.
The restriction map of cDNA is shown.

FIG. 5 shows the restriction map of the E2 fragment of the cDNA derived from HIV-2. This fragment is HI
Includes a region corresponding to the 3'LTR region of V-2.

FIG. 6 shows the nucleotide sequence of a portion of E2. This sequence corresponds to the U3 / R region of HIV-2.

FIG. 7 shows, on the one hand, the structural elements of HIV-1 (FIG. 7A) and HIV-1 3'L.
3 shows the sequences derived from the E2 region of the HIV-2 cDNA aligned with the region containing the TR, -on the other hand in view of the sequences derived from the E2 region of HIV-2 and the large number of deletions and insertions, HI aligned to the array
The common nucleotide present in the corresponding sequence of V-1 is shown (Fig. 7B).

FIG. 8 shows schematically the structures of several clones of phage (clones ROD4, ROD27 and ROD35) modified by several insertions obtained from a cDNA derived from HIV-2. The sequences from ROD4, ROD27 and ROD35 subcloned in plasmid pUC18 are also shown schematically in the figure. Subcloning sequence is the original ROD
4, ROD27 and ROD35 are arranged to correspond to the area.

FIG. 9 (a) Eleven fragments taken from different regions of the complete HIV-1 genome (the fragments are shown schematically at the bottom of the figure) and HIV present in ROD4. Shows the relative intensity of hybridization between -2 cDNA and (b) a fragment obtained from HIV-2 with the same cDNA.

In general, the HI described below used in the comparative tests
The V-2 antigen is HIV-2 M deposited at CNCM with accession number I-502.
The DNA sequence obtained from the IR strain and derived from the HIV-2 genomic DNA is obtained from the HIV-2 ROD strain deposited at the CNCM under accession number I-532.

I. Antigens, especially proteins and glycoproteins For the purpose of metabolically labeling the virus initially cultivated in HUT78 with [ ³⁵ S] cysteine and [ ³⁵ S] methionine, the above radioactive amino acids in medium without the corresponding unlabeled amino acids Infected cells were incubated for 14-16 hours in the presence of For [ ³⁵ S] cysteine labeling, the method described in Reference (21) in the reference list attached to the end of the specification is used. The clear supernatant was then collected and the virus ultracentrifuged at 100,000 g for 1 hour on a buffer of 20% sucrose. The major antigen of the virus was subjected to electrophoresis in a polyacrylamide gel (12.5%) under denaturing conditions (SDS) or a gel consisting of polyacrylamide gel (10%) + bisacrylamide (0.13%) and SDS (final concentration 0.1%). Separated.
The following color markers were used as molecular weight standards.

Myosin 200kd phosphorylase B 97.4kd BSA 68kd ovalbumin 43kd α-chymotrypsin 25.7kd β-lactoglobulin 18.4kd lysozyme 14.3kd In another experiment another molecular weight marker was used. For example, especially in Figures 1a, 1b and 1c (indicated by the letter M in the figure)
Another molecular weight marker was used. Antigens can be readily distinguished after immunoprecipitation (RIPA) or immunoimprinting (Western blot) using antibodies present in the sera of patients. The apparent molecular weight measured by apparent mobility is very close to the molecular weight of HIV-1 antigen.

Generally, in the following description in the text, "p"
And / or the number following "gp" corresponds to the approximate molecular weight of the corresponding protein and / or glycoprotein divided by 1000. For example, p36 has a molecular weight of about 36,000. However, it should be understood that these molecular weight values may vary within the range of 5%, 10% or more depending on the method used to determine the molecular weight.

By repeating the experiment, the apparent molecular weight of HIV-2 antigen could be measured more accurately. Therefore, first about 13,00
The three core proteins, estimated at 0, 18,000 and 25,000 respectively, actually had apparent molecular weights close to 12,000, 16,000 and 26,000. These proteins are referred to hereafter by the abbreviations p12, p16 and p26.

Apparent molecular weight 32,000 to 42,000 to 45,000
The same can be considered for the presence of a protein or glycoprotein band estimated to have values in the range of up to.
By repeating the measurement, the band corresponding to the apparent molecular weight of 36,000 could be accurately defined. This band is indicated by the abbreviation p36 in the following description in the text. 42,000-45,000 (p42)
Another band of is always observed. Perhaps either p36 or p42 constitutes the viral transmembrane protein.

In addition, a major envelope glycoprotein of the order of 130-140 kd is always observed. This glycoprotein is hereafter referred to as gp140.

Generally, the molecular weight is measured with an accuracy of ± 5%, but it should be noted that this accuracy may be somewhat lower for high molecular weight antigens as observed with gp140 (molecular weight 140 ± 10%). When detecting this group of antigens (labeled with [ ³⁵ S] cysteine), in the detection system used in the laboratory, the serum of a patient containing anti-HIV-1 antibody or DIAGNOST
HIV-1 marketed under the trademark "ELAVIA" by ICS PASTEUR
Only weakly recognized by tests using lysates. Only slight p26 protein was immunoprecipitated by this serum. The envelope protein did not sediment.
Sera from patients infected with the new virus (HIV-2) are HIV-1
Faintly recognized the p34 protein. No other HIV-1 protein was recognized in the detection system used.

In contrast, HIV-2 was isolated under similar conditions from a retrovirus recently isolated from the captive macaque of rhesus species under similar conditions and to some extent immunized with an equivalent structural or glycoprotein. It has certain proteins that show biological correlation. This immunological correlation disappears with other proteins and glycoproteins. This retrovirus, which is presumed to be the etiological agent of AIDS in monkeys, was successfully isolated by researchers (references (16)-(1).
8)) and named it STLV-III mac. For convenience, in the following description, this retrovirus is simply referred to as STLV-III (or SIV, which is an abbreviation for "Simian Immunodeficiency Virus").

Another retrovirus was isolated from wild green monkey and named STLV-III _AGM or SIV _AGM . However, in contrast to the virus present in rhesus monkeys, "STLV-II present in African green monkeys
It is believed that " _IAGM " does not induce any AIDS-type illness.

However, the retrovirus STLV-III m
The immunological correlation between structural proteins and glycoproteins of HIV-2 is limited both for ac and for STLV-III _AGM , and thus the relationship between nucleic acid sequences is limited. The difference between retroviruses that can infect humans or monkeys was first confirmed by experiments, and the following facts were revealed.

The HIV-2 virus is described by NLLetvin et al., Scien.
as described by ce (1985), vol.230, 71-75.
STLV-III mac Does not proliferate chronically in rhesus monkey lymphocytes when injected in vivo under conditions capable of proliferating.

Thus, based on the presumption that the HIV-2 virus cannot grow in monkeys under natural conditions, HIV-2
The virus can be biologically distinguished from the STLV-III virus isolate.

Using the method quoted above, the following proteins were obtained from STLV-III.

A major core protein p27 of the order of 27 kilodaltons molecular weight, a major envelope glycoprotein gp140, a pre-labeled virus with [ ³⁵ S] cysteine
Probably the transmembrane protein p32 which is not observed in RIPA but can be seen as a broad band in immunoimprinting (Western blot) studies.

It was found that the HIV-2 major envelope glycoprotein is immunologically closer to the STLV-III major envelope glycoprotein than to the HIV-1 major envelope glycoprotein.

These findings are proved not only from the viewpoint of molecular weight but also from an immunological viewpoint. That is, HIV-2 and STLV
The major glycoprotein of -III has a molecular weight of 130-140 kilodaltons, whereas the major glycoprotein of HIV-1 has a molecular weight of approximately 110 kilodaltons, and sera collected from HIV-2 infected patients. In particular, antibodies raised against HIV-2 gp140 recognize STLV-III gp140 but not HIV-1 gp110.
Do not recognize. However, anti-HIV-1 serum that did not react with HIV-2 gp140 is present in the HIV-2 extract [ ³⁵ S].
Precipitate the cysteine labeled 26kd protein.

The main HIV-2 core protein is HIV-1 p2.
Average molecular weight between 5 and STLV-III p27 (about 26,000)
With.

These observations were obtained by experiments carried out with viral extracts obtained from HIV-2 isolated from one of the said patients. Similar results were obtained with the HIV-2 extract from the second patient.

The cells infected with HIV-1, HIV-2 and STLV-III, respectively, were treated with 200 μCi / ml [unlabeled cysteine-free].
Incubated for 16 hours in medium containing ³⁵ S] cysteine. The clear supernatant was centrifuged at 60,000 g for 90 minutes. The pellet was dissolved in RIPA buffer (1), immunoprecipitated with various sera, and subjected to polyacrylamide gel electrophoresis packed with sodium dodecyl sulfate (SDS-PAGE).

The observed results are shown in FIGS. 1a, 1b and 1c.
Shown in the figure.

FIG. 1a was obtained from the CEM C1.13 cell line
Shown are the observations of immunoprecipitation between a viral extract of HIV-1 and each of the following sera.

-Anti-HIV-1 positive sera (band 1),-serum obtained from said first patient (band 2),-serum of healthy African anti-HIV-1 antibody carrier (band 3),- Sera obtained from Rhesus macaques infected with STLV-III (band 4), and-serum obtained from the second patient mentioned above (band 5).

FIG. 1b shows the relationship between the HUT78 cells collected from the first patient, the HIV-2 antigen precultured, and various sera, namely, the serum of the first patient (band 1), anti-HIV-1 positive. Serum (band 2), STLV-III-infected rhesus macaque serum (band 3) and serum of the second patient (band 4)
The observation result of the immunoprecipitation between and is shown.

Finally, FIG. 1c shows the results of observation of immunoprecipitation between antigens of STLV-III isolates obtained from Macaque Rhesus macaque with AIDS in monkeys. The sera used for bands 1-5 are the same as in Figure 1a.

M represents a marker such as myosin (200 kd), galactosidase (130 kd), bovine serum albumin (69 kd), phosphorylase B (93 kd), egg albumin (46 kd) and carbonic anhydrase (30 kd).

FIGS. 2a and 2b show that HIV-1, HIV-2 and
The comparison result of the electrophoretic mobility of STLV-III protein is shown.

FIG. 2a relates to the experiments carried out after SDS-PAGE immunoprecipitation of virus extracts labeled with [ ³⁵ S] cysteine. The different bands relate to the following viral extracts: Immunized with human negative control sera carrying no virus (band 1), anti-HIV-1 or anti-HIV-2 antibodies, taken from patient 1 and immunoprecipitated by sera from the same patient. STLV immunoprecipitated with the same virus extract that was precipitated (band 2), serum of Rhesus macaques infected with STLV-III
-III virus extract (band 3), immunoprecipitation observed between the same virus extract and negative control serum (band 4), HIV-1 extract immunoprecipitated with sera of European AIDS patients.

FIG. 1b shows the results obtained in the Western blot (immunoimprinting) experiment. Cell lysates of infected or uninfected HUT-78 cells are subjected to SDS-PAGE electrophoresis and electrophoretically transferred to nitrocellulose filters before reaction with the serum of the first patient (100-fold dilution). The nitrocellulose filter is then washed and the bound antibody visualized with ¹²⁵ I-labeled goat anti-human IgG is detected.

The spots observed in bands 1, 2 and 3 are respectively the agglutination reaction between the serum and the extract, ie uninfected.
HUT-78 cell extract (band 1), HIV-2 virus-infected HUT-78 cell extract (band 2), STLV-III-infected HUT-78 cell extract (band 3) Shows the agglutination reaction between. The numbers in the lateral margins of the bands correspond to the approximate molecular weights of most representative viral proteins (molecular weight kilodaltons).

II. Nucleic acid 1) RNA of HIV-2 retrovirus The viral RNA attached to the filter by the "spot blot" method is the DN of HIV-1 under stringent conditions.
Did not hybridize with A.

“Stringent conditions” refers to HIV-2
It means the treatment conditions in which the hybridization reaction between RNA and a selective probe radioactively labeled with 32 P (or labeled by another method) is performed after washing the probe. Hybridization is membrane, especially 50% in 0.1% SDS / 5 x SSC
18 at 42 ° C in the presence of aqueous formamide (v / v)
Done on time. The membrane in which the hybridization reaction has taken place is then washed at 65 ° C. in a buffer containing 0.15% SDS and 0.1 × SSC.

It is to be understood that "non-stringent conditions" means the processing conditions under which the hybridization reaction and washing are carried out. That is, hybridization was induced by contacting with a selective probe labeled with ³² P (or other method) in 5 × SSC buffer containing 0.1% SDS containing 30% formamide at 42 ° C. for 18 hours. 50 ° C in buffer containing SDS and 2xSSC
Wash the membrane with.

In addition, a hybridization probe consisting of recombinant plasmid pBT1 obtained by cloning HIV-1 DNA derived from λJ19 (Cell, 1985, vol.40, p.9) in vector pUC18 was used. Hybridization experiments were performed. HIV-2 under non-stringent conditions
Weak hybridization was observed between the RNA of Escherichia coli and the cloned DNA derived from HIV-1.

Another probe containing the cloned sequence of HIV-1 was also used.

(A) HIV-1 subgenomic DNA produced from a HIV-1 genomic subclone and inserted into phage M13
Single-stranded probe. The cloned region was associated with the protease gene or the endonuclease gene.

Under non-stringent conditions, one probe of the endonuclease region of HIV-1 (bases 3760 and 41
Only nucleotide sequences between 30) gave weak hybridization with HIV-2. "Protease" probe (HIV-1 nucleotide sequence between bases 1680 and 1804)
Did not hybridize with HIV-2 under non-stringent conditions.

(B) The probe pRS3 (which is subcloned in pUC18) consisting of the coding sequence for the HIV-1 "envelope" region is
It did not hybridize to HIV-2.

Spot blotting is also known as dot blotting (spot-to-spot transfer).

HIV-1, HIV-2 and STLV-III genomic RNA
The results of the hybridization between the probe and the probes containing various subgenomic sequences of HIV-1 virus are shown in FIG.

The supernatants of the various media (0.5-1 ml for each spot) were centrifuged for 5 minutes at 45,000 rpm. The pellet was resuspended in NTE buffer containing 0.1% SDS and attached to a nitrocellulose filter. The filter 2
× SSC medium (0.3M NaCl, 0.03M sodium citrate)
It was previously dipped in. After baking (80 ° C for 2 hours), the filters were hybridized with various probes containing the HIV-1 genomic subfragment under non-stringent conditions (30% formamide, 5xSSC, 40 ° C) and
It was washed at 50 ° C with 2xSSC containing% SDS and autoradiographed at -70 ° C for 48 hours with an intensifying screen.

Probes 1-4 are single-stranded probes obtained by the "prime cut" method described in (25). Briefly describing this method, the single-stranded fragment carrying the subgenomic HIV-1 insert (30) from the M13 virus was transformed into M1.
It was ligated to the oligomeric fragment (17 nucleotides) obtained from 3 (BIOLADS). Next, dA labeled with ³² P at α position
In the presence of TP, dGTP, dTTP and dCTB (Amersham, 3000 Ci / mmol), TM buffer (10 mM Tris, pH 7.5, 10 mM MgCl 2
_The complementary strand was synthesized with Klenow enzyme in ₂ ). The DNA was then digested with the appropriate restriction enzymes, heat denatured and electrophoresed on a denaturing polyacrylamide gel (6% acrylamide in TDE buffer containing 8M urea). The gel was then autoradiographed for 5 minutes. Then cut the probe, 300m
Elution with M NaCl, 0.1% SDS buffer. The specific activity (SA) of these single-stranded probes was quantified to obtain 5 × 10 ⁸ -10 ⁹ disintegration amount / minute / microgram (dpm / μg).

The characteristic sequences present in the various probes are shown below.

Probe 1: nucleotide 990-1070, probe 2: nucleotide 980-1260, probe 3: nucleotide 2170-2240, probe 4: nucleotide 3370-3640.

The numbering of the above nucleotides is based on reference (30).

Finally, probe 5 was selected for HIV in λJ19 (31).
It consists of the plasmid pUC18 which carries the EcoRI-SacI fragment of the clone. When this was processed by nick translation, SA of about 10 ⁸ dpm / μg was obtained.

The relative placement of the subgenomic fragment present in the probe relative to complete HIV-1 is shown in FIG. The various spots correspond respectively to:

Spot A: CEM C1.13 infected with HIV-1
Virus obtained from cell culture, spot B: virus obtained from HUT-78 cells infected with STLV-III, spot C and D: single virus obtained from each of the two African patients. Separation, Spot E: Negative control cell extract obtained from uninfected HUT-78 cells, Spot F: Virus taken from Zaire AIDS patient and cultured in normal T lymphocytes in the presence of TCGF.

All spots except spot C were obtained with a viral load corresponding to a reverse transcriptase activity of 25,000 dpm. Spot C corresponds to 15,000 dpm.

The following observations were made:

Although virus particles exhibiting high reverse transcriptase activity were isolated and purified from the culture supernatant of highly infected cells, genomic RNAs of two HIV-2 isolates obtained from the purified virus particles were obtained. Did not hybridize to any probe under the stringent conditions. .

The following observations were made under the non-stringent conditions. All probes control HIV-
1 It hybridized strongly with genomic RNA obtained from the preparation and from another isolate of Zaire AIDS patient.

Two of the probes obtained (both HIV-1
Nucleotides 990-1070 and 990- from the gag region of
1260) hybridized slightly with spots from the HIV-2 retrovirus extract. Only one of these two probes (nucleotides 990-1260) hybridized slightly with the STLV-III spot (Fig. 3). pol
For probes containing a fragment of the region (nucleotides 2170-2240), hybridisation with STLV-III was observed, and to a much lesser extent, hybridisation with RNA of HIV-2. Another probe in the pol region (nucleotides 3370-3640) did not hybridize to either HIV-2 or STLV-III spots.

Finally, the complete env gene of HIV-2 modified by nick translation and the LTR (nucleotides 5290-9
130) and probes include STLV-III RNA and HIV-2 RN.
It did not hybridize with any of A.

Also noteworthy is that another probe containing the 5'end of the pol reading frame of HIV-1 (corresponding to the protease region) also does not hybridize to either HIV-2 RNA or STLV-III RNA. That is.

Therefore, from the above results, it is considered that HIV-2 virus is closer to STLV-III than HIV-1 from a structural point of view. However, HIV-2 is also significantly different from STLV-III, which supports various observations regarding the infectivity of the HIV-2 virus. That is, the HIV-2 virus has virtually no infectivity for monkeys, whereas the STLV-III virus shows a clear infectivity for monkeys of the same species.

The restriction map and sequence of HIV-2 and the genomic RNA sequence or the cDNA obtained from this genomic RNA can be easily deduced by those skilled in the art. This is because the HIV-2 strain deposited at the CNCM provides the genetic material necessary for the determination of these restriction maps and nucleotide sequences after proper amplification. The conditions under which a restriction map of one of the HIV-2 isolates of the present invention is established and the conditions under which the sequences of some parts of the cDNAs derived from these genomes are determined are described below.

FIG. 4 shows a restriction map of the genome of a retrovirus representative of the HIV-2 retrovirus. Figure 5 shows a restriction map of the substantial fragment of this cDNA. Finally, a portion of this fragment was sequenced.

FIG. 6 shows this sequence and the numerous restriction sites it contains. The cloned complete cDNA or the cloned fragment of this cDNA itself can be used as a specific hybridization probe.

2) cDNAs derived from HIV-2 RNA and
Fragments of the cDNA The conditions under which this cDNA is obtained are described below.

In the first step of this cDNA production, HIV-2 reverse transcriptase was used to cause purified virions obtained from the supernatant of infected CEM cells to undergo a detergent-activated endogenous reaction. Oligo that functions as a primer
(dT) or initiator cDNA strand is produced. CEM cell line
GE Foley et al. Cancer 18: 522-529 (1965) lymphoblastoid CD4 + cell line. This document is included in this specification. ROD these used CEM cell lines
It was found that infection with the isolate produced a significant amount of HIV-2 continuously.

After synthesis of the second strand (in the presence of nucleotides and bacterial DNA polymerase), the double-stranded cDNA was inserted into the bacterial phage vector M13 TG130. 10 ⁴ to obtain a phage library of recombinant M13 phage, which was treated with in situ screening with HIV-1 probe. HIV-1
The probe contains a 1.5 kb fragment derived from the 3'end of the cDNA derived from the RNA of the LAV isolate (see Figure 7A). About 50 positive plaques were detected, these were purified and characterized by cross-hybridization of inserts and sequencing of the ends.

By this processing procedure, LTR (S. Wain Hobso
n, et al., Cell 40: 9-17 (1985), `` long terminal re
Various clones were isolated which contained a sequence that was approximately complementary to the 3'end of the polyadenylated RNA in the "abbreviation of peat" region (the above references are included herein).

The largest insert in the group of M13 clones of interest that hybridizes to the 3'LTR region of HIV-1 is the approximately 2 kb clone designated E2. Similar to the HIV-1 3'LTR clone, clone E2 contains the AATAAA signal located approximately 20 nucleotides upstream from the poly (A) end and the 3'LTR region corresponding to the HIV-2 3'LTR region. Including and After partial sequencing, this 3'LTR region of HIV-2
It was found that it is not related to the homology region of V-1.

FIG. 5 was integrated into the plasmid pSPE2
A restriction map of an E2 fragment (elongated rectangular region) is shown. This includes the R region part of HIV-2 and the U3 region. The figure is
It does not show the boundary between the R area and the U3 area.

The sequence of the E2 portion is shown in FIG. Figure 6 also shows the location of specific restriction sites. Between HIV-1 and HIV-2
It is shown in Figure 7 that the 3'LTR regions are not closely related to each other. In fact, only about 50% of the two LTR sequences are aligned with some insertions and deletions (about 50%).
Sequence homology). In contrast, the corresponding regions of various isolates of American and African HIV-1 show greater than 95% sequence homology without insertions or deletions.

Clone E2 was used as a specific probe for HIV-2 and the sequences derived from HIV-2 present in another clone were identified on a hybridization filter.

This probe also detects HIV-2 genomic RNA under stringent conditions. This probe is also capable of detecting the DNA of CEM or similar cells infected with ROD isolates or another HIV-2 isolate by the so-called Southern blot technique. No signal was detected in the hybridization test of cDNA from non-infected cells or HIV-1-infected cells with this probe under the same stringent conditions. These results confirmed that HIV-2 is exogenous compared to HIV-1. Virus DN not included
A species of approximately 10 kb presumed to correspond to A was detected as a major component of undigested DNA in HIV-2 infected cells. Viral DNA
Another DNA with an apparent size of 6 kb, which is supposed to correspond to the circle of, was also detected.

Another portion of the HIV-2 genome was also identified. For this, a genomic library was constructed in the phage lambda L47. Phage Lambda L47.1 is WAMLoenen et al.
Gene10 249-259 (1980). This reference is incorporated herein.

A genomic library was constructed with the fragments obtained by digesting DNA from the CEM cell line infected with HIV-2 ROD after digestion with the enzyme Sau3A I.

Approximately 2 × 10 ⁶ recombinant plaques were screened in situ with clones containing the labeled E2 insert of HIV-2 cDNA. Ten recombinant phage were detected and purified on plaques. Restriction maps of three of these phages were determined by Southern blot hybridization with the E2 insert under stringent conditions and Southern blot hybridization with the HIV-1 subgenomic probe under non-stringent conditions. Characterized by

Whole Circular Virus DN Containing the Complete HIV-2 Genome
A clone derived from A and containing a 9.5 kb insert was identified. This was named "Lambda ROD4". Two other clones derived from the integrated provirus, Lambda ROD27 and Lambda ROD35, are the LTRs of the viral coding sequence.
It carries the sequence and the flanking cell DNA sequence. The sequence of each clone is shown in FIG.

A fragment of the lambda clone was subcloned in the plasmid vector pUC18. λROD4,
The fragments of λROD27 and λROD35 and the respective subclones in the above plasmid vector are also shown in FIG. The following subclones were obtained.

PROD27-5 'from lambda ROD27 containing the 5.2 kb region of the HIV-2 genome and flanking cellular sequences (5'LTR and 5'coding viral sequences surrounding the EcoRI site).

PROD4.8 from lambda ROD4 containing a HindIII fragment of approximately 5 kb. This fragment is HIV-2
It corresponds to the central part of the genome.

-PROD27-5 'and pROD4.8 containing HIV-2 inserts that overlap each other.

PROD4.7 containing the 1.8 kb HindIII fragment of lambda ROD4. This fragment is located in three orientations with respect to the subcloned fragment in pROD4.8 and contains approximately 0.8 kb of the coding viral sequence and BamHI and Hind in the left arm of phage lambda (lambda L47.1).
And the portion existing between the cloning sites of III.

PROD35 containing the entire HIV-2 coding sequence in 3 orientations to the EcoRI site, the 3'LTR and approximately 4 kb of contiguous nucleotide sequence of cellular origin.

PROD27-5 ′ present in E. coli HB101 and
pROD35 was deposited at the CNCM on November 21, 1986, with deposit numbers I-626 and I-633, respectively.

PROD4.7 and pROD present in E. coli TG1
4.8 was assigned to CNCM on 21st November 1986 with accession numbers I-627 and
Deposited on I-628.

Complete HIV-2 whose restriction map is shown in FIG.
The ROD genome was pre-linearized with EcoRI, pROD35 and pROD27.
-Reconstructed from 5 '. pROD27-5 ′ EcoRI insert into pR
It bound to the EcoRI site of OD35 in the correct orientation.

The degree of correlation between HIV-2 and other human or simian retroviruses was assayed by a mutual hybridization test. The relative homology between different regions of the HIV-1 and HIV-2 genomes was determined by hybridization studies of fragments from the cloned HIV-1 genome with those from the radiolabeled Lambda ROD4. The relative positions of these fragments (numbers 1-11) with respect to the HIV-1 genome are shown at the bottom of FIG.

Under extremely weak stringent conditions (42
Even the HIV-1 and HIV-2 genomes have their own gag
Gene (spots 1 and 2), pol reverse transcriptase region (spot 3), pol end region, Q (or sor) gene (spot 5) and F (or 3'orf) gene and
Only hybridized at the level of 3'LTR (Spot 11). Used to detect the first HIV-2 cDNA clone
The HIV-1 fragment corresponds to the spot 11 subclone and hybridizes relatively well with HIV-2 under non-stringent conditions. The signal obtained from spot 5 is the only signal that survives stringent washes. The envelope gene, tat gene region, and pol part seem to vary widely. The sequence obtained from these data and LTR (Fig. 3) is HIV-2.
Is not a variant of HIV-1 (at least with respect to its envelope).

HIV-2 is more closely related to SIV than HIV-1 (MD Daniel et al., Science 228: 1201-1204 (1985), which is incorporated herein by reference).

All SIs including envelope proteins
The V protein is immunoprecipitated by sera from HIV-2 infected patients, but serological cross-reactivity between HIV-1 and HIV-2 is restricted to the core protein. However, with SIV
As mentioned above, HIV-2 can be identified by the difference in the molecular weight of each protein.

With respect to nucleotide sequence, HIV-2 is also closely related to SIV.

Furthermore, characterization of HIV-2 makes it possible to define the regions of the envelope glycoprotein which induce the binding of the virus to the surface of the target cell and the internalization of the virus. The interaction occurs via the CD4 molecule itself. It is believed that HIV-1 and HIV-2 use the same receptors.

Thus, although there are large differences between the env genes of HIV-1 and HIV-2, the restricted homologous region of the envelope of these two forms of HIV is
It can be considered to be a component that binds to the common receptor of lymphocytes. The action of these sites forms epitopes that carry the immunogenicity of peptides that elicit a protective immune response in humans against the HIV virus.

Sequences that are advantageous for forming probes in the hybridization reaction with viral or proviral carrier genetic material, and particularly for detecting the presence of HIV-2 viral RNA in lymphocytes, include ROD4 and E2 cDNA. With 5k
It contains the nucleotide sequence obtained by ligation of the Hind fragment of b. The experiment may be performed by any method, in particular Northern blot, Southern blot and dot blot methods may be used.

Furthermore, the identification of several parts of the retroviral genome and the derivation of the retroviral genome of HIV-2
The features of the invention will be further clarified by the following description of an example of the production of a large number of recombinant DNAs containing different parts of the cDNA. However, the present invention is not limited to the description of these examples.

[0133]

Example I DNA probe used in HIV-2 diagnostic kit cDNA complementary to genomic RNA obtained from purified virions was prepared by the following method.

CEM infected with HIV-2 ROD isolate of HIV-2
The supernatant obtained after culturing the cells for 48 hours was ultracentrifuged.
Centrifugal pellets containing virions were centrifuged on a sucrose gradient to form new centrifugal pellets, using substantially the same method as described in European Patent Application No. 84 / 401.234-0138667, supra.

Purified HIV-2 preparations were used to synthesize cDNA using detergent-activated endogenous reactions.

In summary, the virion preparation was prepared by adding 50 mM Tris-HCl, 5 mM MgCl ₂ , 10 mM DTT, 0.025% Triton detergent (trade name TRITON) and 50 μM each of four deoxynucleoside triphosphates and oligo (dT). ) Was added to the reaction mixture containing the initiator. The reaction was held at 37 ° C for 90 minutes.

The protein present in the first reaction medium was extracted with phenol, and RNase and E. coli DNA polymerase were extracted.
At 15 ° C in the presence of 1 and 4 deoxynucleotides
The second DNA strand was synthesized by maintaining it for 1 hour and at 22 ° C for 1 hour. This double-stranded cDNA was treated with T4 DNA polymerase to form blunt ends. All reagents used in this procedure were commercially available (AMERSHAM cDNA kit) and used according to manufacturer's instructions.

(1) T4 DNA ligase (BIOLABS commercial product)
Adapter (linker) (Ph
armacia commercial product) to the blunt ends of the cDNA,
These linkers were digested with the restriction endonuclease EcoRI and gel-filtered on (3) AcA 34 (LKB-IBF) (trade name ULTROG).
The linker fragment was removed by EL gel column) and the cDNA was inserted into the M13TG130 vector cleaved with Eco RI.
Insert. After transformation of E. coli TG1 strain, a cDNA library was obtained. About 10 ⁴ recombinant M13 plaques were obtained.

The plaque hybridization method was used to select recombinant M13 clones containing the HIV-2 cDNA from the cDNA library. Transfer the DNA of M13 plaques to nitrocellulose filters, and
It hybridized with a subgenomic HIV-1 probe derived from the "lambda J19" clone of the LAV (or HIV) virus. This probe contained an insert consisting of an approximately 1500 base pair (bp) long portion of HIV-1 DNA. This insert was joined by two Hind III restriction sites within the open frame of the env gene and within the R segment of the HIV-1 3'LTR. This probe contained the 3'end of the env gene, the complete F gene, the U3 segment and part of the R segment of the LTR, and was about 1500 base pairs (bp) long.

A probe containing a 1.5 kb HindIII insert was used (using the AMERSHAM kit) with an initiator and Klen.
ow DNA polymerase I for 4 hours at 15 ° C to induce [ ³² P-] dCTP and -dTTP (30
00Ci × 10 ⁻³ mol). Hybridization studies of the cDNA clones of the library were performed overnight under mildly stringent conditions. The hybridization medium solution used was 5 x SSC and 5 x Denhart 25 ° C at 37 ° C.
% Formamide, 100 μg / ml denatured salmon sperm DNA and labeled probe (specificity 10 ⁹ cpm / μg at 2 x 10 ⁷ cpm).
) Was included.

The filter was washed in three stages by sequentially using three kinds of solutions having the following compositions.

First stage wash: 5 × SSC, 0.1% SDS, 25 ° C., 4 × 15 minutes Second stage wash: 2 × SSC, 0.1% SDS, 42 ° C., 2 × 30 minutes Third stage wash: 0.1 × SSC, 0.1% SDS, 65 ° C, 2 x 30 minutes.

Filters were autoradiographed after each wash step.

Multiple positive clones were detected after the first wash and were still detected after the second wash. However, all signal disappeared after the third wash. This indicates that the positive clones are not closely related to the HIV-1 genome, but are nevertheless sufficient to carry out the selection. Positive clones were subcultured, reattached to plaques, and rehybridized with the same probe under the same stringent conditions as in the first step washing. Most of these clones were still positive.

Using fully human DNA probe under moderately stringent conditions, 5 × SSC, 5 × Denhart
And in 40% formamide, then at 50 ° C.
Clones were selected by washing with 1 × SSC, 0.1% SDS. No positive clones were detected previously and therefore did not correspond to the specific repetitive DNA or the cDNA of ribosomal RNA.

Positive M13 recombinant clones were cultured in liquid medium and characterized as follows.

(1) Size of insert M13 single-strand type DNA was extracted from each clone and
Second using the 3 17-mer initiator sequence and Klenow enzyme
A chain was formed. The insert was excised with Eco RI (BOEHRINGER) and analyzed by agarose gel electrophoresis. 2 for most inserts
It contained 00-600 and 200 bp, except clone E2.1, which was approximately 2 kbp in length.

(2) Analysis of nucleotide sequence Sanger described in Proc. Natl. Acad. Sci. 74: 5463-7 (1977).
Multiple clones were partially sequenced using the dideoxy method of et al. This document is included in this specification. Various independent clones contained similar nucleotide sequences, with the exception of the poly (A) chain at the 3'end, which had different lengths. These results indicate that these cDNA clones were derived from RNA templates. Detailed sequence analysis revealed that the cDNA clone containing the 3'end of the HIV-2 genome was
It turns out that it is not related to HIV-1.

(3) High level of HIV-2 genomic RNA and DNA
Hybridization (a) Production of HIV-2 genomic RNA The infection supernatant was centrifuged (50,000 rpm, 30 minutes). The pellet of pellet was resuspended in 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS. One of the insert clones, F1.1, was labeled and used as a hybridization probe with genomic RNA from various viral isolates by dot blotting.

The dot blot method comprises the following steps.

(I) A spot of the sample (HIV-2 solution) was attached to a nitrocellulose membrane pre-soaked in 20 × SSC (3M NaCl, 0.3 sodium citrate) and air-dried,
(Ii) The membrane is baked at 80 ° C for 2 hours, and (iii) hybridization is performed.

This hybridization was performed under strongly stringent conditions (5 × SSC, 5 × Denhart).
, 50% formamide, 42 ° C). Then 0.1 × SSC, 0.1
% SDS, washed at 65 ° C. Under these conditions, the probe hybridized vigorously with spots of two separate HIV-2 isolates containing LAV-II ROD as the origin of the cloned cDNA. STLV-III mac (monkey T lymphotropic virus
(Also known as SIV), Type III Rhesus macaque
(macaque)), a weak hybridization signal was detected in the spots, and no hybridization was detected in the HIV-1 isolate.

When Southern blot experiments were performed using clone E2.1 containing the 2 kb insert as a ³² P-labeled probe, no hybridization between this clone and the DNA of uninfected cells was detected, although strong stringency was detected. Bands were detected in isolated cells infected with HIV-2 under mild conditions. HIV-2 exhibits a polymorphism equivalent to HIV-1 at the restriction map level. Two types of signals are detected by Southern blotting in the intact cell DNA of infected cells. That is, (1) the molecular weight MW for the integrated form of the virus.
Signal is detected in the DNA fraction of about 20 kb or more, (2)
For viruses that do not integrate into the genome, low molecular weight (9,1
A signal is detected in the 0 kb fraction.

These properties are highly specific to retroviruses.

Several experiments carried out with STLV-III (SIV-3) in infected cells showed that the monkey retrovirus was HIV-
It was confirmed that it was not closely related to 2 (the signal can only be detected under weakly stringent conditions).
These experiments show that the probe is capable of specifically detecting HIV-2.

(4) HIV-2 in a bacterial plasmid vector
CDNA subcloning positive M13 clone E2.1 was selected and subcloned in a plasmid vector. Recombinant M13 (TG130) phage E2
DNA was single-stranded DNA (M-13-ROD-E2) containing a 2 kb insert containing the 3'part of the HIV-2 genome (obtained from the HIV-2 ROD).
It was purified in the form of Insert this insert with Melton, DA, 357 Nu
cleic Acid Res. 12: 035-7056 (1984).

17-mer initiator sequence (AMERSHAM) and 4
Nucleotides A, C, T, G and DNA polymerase I (Kleno
The second strand was constructed in vitro in the presence of w). The Eco RI insert was excised by Eco RI digestion, purified on an agarose gel and ligated to pSP65 pre-digested with Eco RI. The ligation mixture was used to transform E. coli strain DH1 and recombinants selected based on ampicillin resistance. LB medium (Luria medium) containing 50 μg / ml ampicillin of the identified recombinants
It was cultured in. These plasmids were purified and monitored for the presence of correctly inserted fragments.

One of the resulting clones was designated pSPE2 and was deposited on September 5, 1986 with the CNCM in Paris, France under accession number I-595.

The insert derived from the HIV-2 cDNA and inserted into the probe contained a nucleotide sequence as defined above which corresponds to a portion of E2.

EXAMPLE II HIV-2 Virion Genomic RNA Complementary to DNA Complementary to DNA
cDNA cloning 5 l of cell supernatant from CEM strain infected with ROD isolates from HI
The V-2 virion was purified. The first strand of the cDNA was synthesized using a detergent-activated endogenous reaction in contact with precipitated purified virus in the presence of oligo (dT) initiator. For this, the method of Alizon et al., Nature 312 , 757-760 (1984) was used. The RNA / cDNA hybrid was purified by extraction with a phenol / chloroform mixture and precipitation with ethanol. The second strand of the cDNA was prepared by the method of Gubler and Hoffman.
Produced in the presence of DNA polymerase I / RNase H.
The description of the document is included in the present specification.

Blunt ends were formed on the double-stranded cDNA in the presence of DNA polymerase T4 using the components of a commercial cDNA synthesis kit from AMERSHAM.

A commercially available EcoRI adapter (linker) from PHARMACIA was attached to the ends of the cDNA. The cDNA modified in this way was also digested with AMRI after digestion in the presence of EcoRI.
Dephosphorylated phage vector M13tg130 marketed by ERSHAM and predigested with EcoRI itself
Was inserted into. A cDNA band was obtained after transformation of the E. coli TG1 strain. The above 1.5 kb HindII obtained from HIV-1
Recombinant plaques (10 ⁴ ) were screened in situ with a filter that was able to replicate by hybridization with clone J19 containing the I fragment.

The filter was set with 5 × SSC, 5 × DENHARDT solution, 25% formaldehyde and denatured salmon sperm DNA (100 μm).
(g / ml) in the presence of medium at 37 ° C for 4 hours, and then in the same buffer (Tm-42 ° C) for 16 hours in the presence of additional labeled probe (4 x 10 ⁷ cpm). A final hybridization buffer solution containing 10 ⁶ cpm / ml was obtained.

Washed with 5 × SSC, 0.1% SDS solution for 2 hours at 25 ° C. (it will be understood that 20 × SSC corresponds to a solution of 3M NaCl and 0.3M sodium citrate). The plaques that reacted positively were purified, and M13 single-stranded DNA was prepared.
The ends were sequenced by the method of Nger et al.

DNA Levels of Cells Infected with HIV-1 and HIV-2
And HIV-1, HIV-2 and SIV virion RNAs and HIV-2
Hybridized with a probe derived from the cloned cDNA of
DNA was extracted from infected CEM cells producing homogenization HIV-1 and HIV-2 respectively continuously. Of these two types of retroviruses
DNA samples were digested with 20 μg Pst I in some cases and undigested in others, subjected to 0.8% agarose gel electrophoresis and transferred to nylon membranes by the Southern method. 0.1
A small amount of infection supernatant taken in NTE buffer with the same reverse transcriptase activity containing% SDS was attached to nitrocellulose presoaked in 2xSSC solution.

50% formamide, 5 × SSC and 5 × Denh
in a solution containing art and 100 mg / ml denatured salmon sperm DNA
Prehybridization was performed at 42 ° C for 4 hours.
10% dextran sulfate and 10 ⁶ cpm / ml in the same buffer
Hybridization was carried out at 42 ° C. for 16 hours in a solution to which an E2-labeled insert (specific activity 10 ⁹ cpm / μg) was added.
Next, 0.1 × SSC and 0.1% SDS solutions were used and washed twice for 30 minutes each. Exposure to intensifying screen for 16 hours, 0.4N
Southern spots were dehybridized in NaOH, neutralized and rehybridized under the same conditions with an HIV-1 probe labeled with 10 ⁹ cpm / μg.

EXAMPLE III Complete DNA of HIV-2 Provirus in Phage Lambda
CEM infected with cloned HIV-2 ROD (Fig. 2, bands a and c)
The DNA of the cells partially digested with Sau 3AI. Fractions 9~15kb selected in 5 -40% sucrose gradient and ligated to the Bam HI arm of the lambda L47.1 vector. Plaques (2 × 10 ⁶ ) obtained after in vitro packaging and attachment to E. coli strain LA101 were screened in situ by hybridization with the insert of the E2 cloned cDNA. About 10 positive clones were purified on plaques and grown on E. coli C600 recBC. Clone lambda ROD4, ROD2
7 and ROD35 were amplified. Create each restriction map,
Hybridization was carried out under stringent conditions with the HIV-2 cDNA clone by the Southern method.
DNA was characterized by hybridization under non-stringent conditions with the gag-pol probe.

FIG. 8 shows the three recombinant phages RO obtained.
The structures of D4, ROD27 and ROD35 are shown schematically.

The elongated rectangles in these figures correspond to the proviral sequences obtained from the DNA of the originally infected CEM cells. White areas correspond to retroviral sequences.
The shaded area corresponds to the cellular DNA. The black part corresponds to the LTR in the virus sequence.

The thin lines indicated by the letters L and R correspond to the arms emerging from the Lambda L47.1 phage vector used for cloning. Some restriction sites are also shown.
Especially B is Bam HI, E is Eco RI, H is Hind III, K is Kpn
I, Ps is Pst I, Pv is Pvu I, S is Sac I, X is Xba I
It is a part.

These viral sequences have an intersection with the E2 sequence. Also shown are the relative positions of these moieties as determined by hybridization with E2.

ROD4 is derived from circular viral DNA.
ROD27 and ROD35 are derived from a provirus integrated in the cellular DNA structure.

Finally, the insert subcloned under the above conditions and the relative position of the insert with respect to the corresponding ROD4, ROD27 and ROD35 sequences are also illustrated.

The insert shown is especially the plasmid pROD27-
5 ', pROD35-3', pPOD4.6, pROD4.8 and pROD4.7 inserts.

FIG. 9 shows subfragments 1-11, respectively, obtained from different parts of the single-stranded DNA obtained from the M13 subclone containing the nucleic acid derived from the complete LAV genome.
The relative intensity of hybridization spots generated with ROD4 are shown. The relative position of these various fragments relative to the complete LAV genome (determined based on sequencing) is shown at the bottom of the figure. Spot 12 corresponds to the control spot produced with the phage lambda control DNA.

Hybridization experiments by the spot transfer (dot blot) method were carried out under the weakly stringent condition of Example II using a lambda ROD4 recombinant containing the complete cDNA of HIV-2 as a probe.
Next, it was sequentially washed under the following conditions. 2 x SSC, 0.1% SDS
, 25 ℃ (Tm-42 ℃), 1 × SSC, 0.1% SDS, 60 ℃ (Tm-2
0 ° C), 0.1% x SSC, 0.1% SDS, 60 ° C (Tm-3 ° C).

The spots shown were obtained after overnight radiograph exposure.

Example IV In Vitro Diagnostic Test for Presence of HIV-2 Virus in Biological Media
Materials and Methods Subjects HIV-2 infected patients were selected from inpatients or outpatients between September 1985 and September 1986 at Egas Moniz Hospital in Lisbon. For this selection, the sera of all Africans or those who have lived in Africa were tested for antibodies to both HIV-1 (immunofluorescence-IFA- or ELISA) and HIV-2 (IFA). The following test was conducted only on subjects with a HIV-2 infection serologically detected.

Virus Isolation HIV isolation was performed as described above on 12 subjects. That is, peripheral blood lymphocytes (PBL) of a subject were stimulated with PHA, co-cultured with normal human PHA-stimulated PBL, and maintained in the presence of interleukin-2 (IL-2). The presence of cytotoxicity of the culture and the reverse transcriptase activity (RT) in the supernatant was monitored.

Immunofluorescence Assay (IFA) IFA slides were prepared as follows. HIV-2 infected
MOLT-4 cells were washed twice with PBS, layered on IFA glass slides (10 ⁴ cells / well), air dried and fixed with cold acetone. These cells were reacted with 10-fold diluted test serum for IFA for 45 minutes at 37 ° C, washed, dried, and fluorescein-conjugated goat anti-human IgG, A, M (100-fold diluted).
And reacted at 37 degrees for 30 minutes. After washing the cells,
Counterstained with Evans blue, 90% glycerol, 10% PBS
It was sealed inside and observed with a fluorescence microscope.

ELISA Commercially available test serum ELAVIA (Pasteur Diagnostics) or ABBO
The TT was used to test the sera of several subjects for HIV-1 antibodies.

Radioimmunoprecipitation Assay (RIPA) CEM cells infected with HIV-1 or HIV-2 were treated with ³⁵ S cysteine.
The cells were cultured in the presence of (200 μCi / ml) for 16 hours. The supernatant was collected, the virus particles were pelleted, and RIPA buffer (Tris-HCl 50 mM pH7.5, NaCl 150 mM, EDTA 1 mM, 1% Triton X100, sodium deoxycholate 0.1%, SDS was added.
0.1%). For each reaction, 50 μl of lysate at a dilution corresponding to 10 ⁵ cpm was added to 5 μl of test serum at 18
Reacted for hours. Immune Complex Sepharose-Protein A
(PHARMACIA), washed and boiled for 2 minutes to elute. The eluted antigen was then analyzed by SDS polyacrylamide gel electrophoresis and autoradiography.

Dot Blot Hybridization The virus isolated from the PBL of the subject was pelleted, Tris-HCl 10 mM pH 7.5, NaCl 10 m, EDTA 1 mM, SDS 0.5.
% Lysed. Each lysate for RT activity of 50,000 cpm
1 μl was attached to nitrocellulose. Hybridization and washing were performed under strong stringent conditions. Hybridization is performed in 6 x SSC, 5 x De
nhart, 50% formamide, 42 ° C for 18 hours, wash at 65 ° C in 0.1X SSC, 0.1% SDS. HIV-1 and HIV-2 probes labeled with ³² P at a specific activity of 10 ⁸ cpm / μg were used. The HIV-1 probe corresponded to the complete genome of the LAV _BRU isolate and the HIV-2 probe was derived from the 2 kb cDNA from the LAV-2 _ROD isolate.

Results Subjects were tested on 30 subjects with serological and / or virological signs of HIV-2 infection. Twelve were men and 18 were women. The ages are 11 to 55, with an average age of 35.
All but one have had several years in West Africa. 26 are Guinea-Bissau residents and 2 are from Cape Verde Islands. One is an 11-year-old boy from Angola who lived on Cape Verde Island for several years. The only Westerner in the trial population is a 40-year-old Portuguese man who has lived in Zaire for eight years and has never stayed in West Africa.

Clinical Findings 17 out of 30 had AIDS according to CDC criteria. The main symptom of these patients was chronic diarrhea, often accompanied by a weight loss of more than 10 kg per year. Diarrhea was associated with intestinal opportunistic infections in 10 patients, in 7 patients the only pathogen was warsp. Isospora belli, in 1 Cryptosporidium alone, and 2 had both pathogens. No intestinal opportunistic pathogens were detected in 3 patients. Esophageal candidiasis was diagnosed in 8 of 17 AIDS patients. Six AIDS patients presented with respiratory syndrome. Two were diagnosed with pulmonary tuberculosis and one had another, unidentified mycobacterium.
Two patients had pulmonary asperogillosis and one had tuberculosis. Two other AIDS patients had repeated episodes of pneumonitis with unidentified pathogens, and one patient had carini pneumocystis (Pneumitis).
ocystis) pneumonia. Only this was found at postmortem diagnosis. 4 out of 17 AIDS patients have Kaposi's sarcoma
In three, this was only on the skin, but in one patient, postmortem findings revealed a scattered visceral lesion. Central nervous system disorders were observed in two AIDS patients. One has cerebral lymphoma and one has subacute encephalitis of unknown cause.

Four patients showed signs of AIDS-related syndrome (ARC). 2 had disseminated lymph node disorders with persistent fever, 1 had chronic diarrhea with weight loss, 1
The person had a recurrent episode of bronchial pneumonia and multiple lymph node disorders.

Of the remaining 9 patients, 6 had no symptoms that seemed to be associated with HIV infection, 1 had only pulmonary tuberculosis,
One had persistent disseminated lymphadenopathy and one had syphilis. Seven patients died during the 12-month study period, all of whom died of AIDS.

Serological Studies Each patient was subjected to at least one serum test for both HIV-1 and HIV-2 antibodies. IFA for all patient sera
All were positive when tested for HIV-2 antibodies. Twenty-one of them were tested for HIV-2 antibody by RIPA.
All sera clearly precipitated the high molecular weight envelope glycoprotein of the virus designated gp140. Sera from 16 of them also reacted with the major core protein p26, and only one serum reacted with another viral core protein designated p16.

Cross-reactivity of sera using various assays
The presence of HIV-1 antibody was tested. IFA tests were performed on 24 sera. Twelve sera were negative, 10 were weakly reactive and 2 were positive. Twenty-one sera were tested by ELISA. Sixteen sera were negative and five sera were positive. Finally, an antibody test for HIV-1 protein in 11 sera was performed with RIPA. Three sera did not react at all with viral proteins, only two sera precipitated the pol gene product p34, and five sera had the major core protein p2.
Reacted with 5. Two sera reacted weakly with the HIV-1 envelope glycoprotein gp110. These two sera and all sera that tested positive for HIV-1 antibodies in IFA or ELISA had strong reactivity with HIV-2 gp140 in RIPA, which is
It is an indicator that there is HIV-2 but not HIV-1 infection. Only one patient who was not included in the test population was serologically HIV-1
And was not infected with HIV-2. The patient is a 21-year-old female AIDS patient from Central Africa (San Tome Island).

Virus Isolation Retroviral isolation studies were performed on peripheral blood lymphocytes in 12 patients. HIV was isolated from 11 samples according to the presence of typical cytopathic effects and the presence of a peak of particle-related reverse transcriptase activity in the culture supernatant.

All 11 isolates were identified as HIV-2 by the dot blot hybridization method. Viral dots of 10 isolates were cloned by hybridization under stringent conditions and washing.
It was found to hybridize strongly with an HIV-2 probe derived from the HIV-2 cDNA, but under the same conditions HIV-1
It did not hybridize to the probe at all. Only one isolate hybridizes weakly with the HIV-2 probe and
It did not hybridize with 1.

Immunological Testing The number of circulating lymphocytes identified as helper T cells (CD4 +) was determined in 13 patients and the ratio of helper T cells to suppressor T cells was calculated. 11 of these patients
The person was AIDS. That is, the average absolute count of helper T cells was 243 + 300 / μl, and the average helper to suppressor ratio was 0.25 + 0.15. Helper T lymphocyte count is 240 / μl in one patient with clinical ARC
The ratio was 0.18. Another patient with cerebral syphilis and no evidence of HIV-related syndrome had a helper T lymphocyte count of 690 / μl with a ratio of 0.82.

Discussion This study demonstrated HIV-2 infection in 30 West African subjects who were afflicted with AIDS or ARC or had no apparent HIV-related syndrome. The conclusion drawn from this result is that HIV-2 is considered to be the major etiological agent in West African AIDS patients. According to the serological and virological profiles observed in our patients,
HIV-2 infection was often not associated with HIV-1 infection. Despite the large antigenic and genetic differences between HIV-1 and HIV-2, they have similar CD4 + T lymphocyte affinity, similar cytopathic effects, similar morphology, and some Have common immunoreactive epitopes in their constituent proteins. The study found that all West African subjects who were HIV-infected were HIV-2 infected and none were HIV-1 infected. Therefore, the novel virus HIV-2 may be a major cause of AIDS in West Africa.

The HIV-2 related AIDS syndrome was not different from the HIV-1 related AIDS syndrome in Central Africa. The most common symptom is chronic diarrhea with considerable weight loss, many of which are war isospora and / or Cryptosporid.
Due to ium. Other opportunistic infections, such as candidiasis, mycobacterium (including M. tuberculosis) and toxoplasmosis, were similar to HIV-1 associated African AIDS. Only one case of Carini pneumocystis pneumonia, the most common complication among Western AIDS patients, was observed during the study and no cryptococcal meningitis was detected.

However, the immunological abnormalities observed in HIV-2 infected AIDS patients are similar to those for HIV-1 related AIDS.

Of all 30 subjects who had serum antibodies reactive with the HIV-2 antigen, only 7 had IFA or ELISA.
It had an HIV-1 specific antibody that could be detected by. In RIPA, all seven subjects had another major core protein, p25 (HIV-1) or p26 (HIV-), that shared a strong immune response epitope.
It had an antibody reactive with 2). Five subjects did not have such antibodies, all five were HIV-1-negative by ELISA, and IFA was three borderline-negative by two. 9 subjects were not fully assayed, but 9 subjects had HIV-1-specific IFA and / or ELIS
It showed weak reactivity to A or was negative, and had a serum antibody against the viral core protein p26 of HIV-2. These findings apply to HIV used in Africa and other regions.
We show that it is important to include the HIV-2 antigen in serum tests.

Retroviruses were isolated from peripheral lymphocytes of 11 patients. In all cases, the virus was grown within 2 weeks and characterized by the presence of reverse transcriptase activity and the cytopathic effect in the culture supernatant. However, this cytopathic effect varies considerably from isolate sample to isolate sample. A large number of large fused cells were formed in some isolates with considerable cell lysis, and a small number of small fused cells were formed in other isolates with little effect on the viability of the culture.

RNA from all but one isolate
Was found to hybridize clearly under strong stringent conditions with a probe derived from an HIV-2 cDNA clone displaying the 3'end of the genome. These isolates did not hybridize to the HIV-1 probe at all under the same conditions. This proves that all isolates infecting these subjects belong to the same type of virus. Only one isolate hybridized with HIV-1 very weakly. However, this virus is
Isolated from patients with serum antibodies that react with all two antigens.

The present invention further includes not only the HIV-2 virus and mutants thereof, but also all equivalent viruses in general that infect humans with immunological properties specific for HIV-2.
The present invention includes not only the characteristics of the HIV-2 virus deposited in the CNCM but also all viruses having the following characteristics.

The preferred target for the HIV-2 retrovirus is human
It consists of Leu3 cells (or T4 lymphocytes) and “immortalized” cells derived from these T4 lymphocytes, for example cells of the HUT78 line described herein above. In other words, the retrovirus has a specific affinity for these cells. The retrovirus is cultivated in HUT, CEM, MOLT or a permanent strain of the same type expressing the T4 protein. The retrovirus does not infect T8 lymphocytes. The retrovirus is cytotoxic to human T4 lymphocytes. T4
The cytopathic effect of HIV-2 virus on lymphocytes is evidenced especially by the appearance of multinucleated cells. The retrovirus has a reverse transcriptase activity that requires the presence of Mg ²⁺ ions and is polyadenylate oligodeoxythymidylate (poly (A)-
It has a strong affinity for oligo (dT), 12-18). The retrovirus has a density of about 1.16 in the sucrose gradient.
The retrovirus has an average diameter of about 140 nm and a core average diameter of about 41 nm. The lysate of this virus contains a p26 protein that does not immunocrossover with the p24 protein of the HTLV-1 or HTLV-2 viruses. Therefore, the molecular weights of these p26 proteins are slightly larger (by about 1,000) than the corresponding p25 proteins of HIV-1 and that of SIV.
Slightly smaller than the molecular weight of p27 protein (about 1,000). The HIV-2 lysate is further processed by RIPA (radioimm
Abbreviation for unoprecipitation assay) HTLV-1 or HTLV in the experiment
Includes p16 protein that is not immunorecognized by p2 protein of -2. These also have a molecular weight of 130,000-140,00.
It contains an envelope glycoprotein of the order 0, which does not immunocross-link with HIV-1 gp110, but STLV-I
It immunologically crosses the gp140 envelope glycoprotein of II.
The lysate also contains proteins or glycoproteins having molecular weights on the order of 36,000 and 42,000-45,000, respectively, which can be labeled with (35S) cysteine. HIV-2 genomic RNA
Does not hybridize to HIV-1 genomic RNA under stringent conditions. The RNA is derived from HIV-1 and is env
It does not hybridize with a nucleotide sequence containing a gene and an LTR adjacent to the gene under non-stringent conditions. The RNA is especially the HIV-1 nucleotide sequence 5290-913.
0 and does not hybridize to any of the sequences of the pol region of the HIV-1 genome, especially the nucleotide sequences 2170-2240. The RNA hybridizes weakly with the nucleotide sequence of the HIV-1 region, in particular the nucleotide sequences 990-1070 and 990-1260 under non-stringent conditions.

Having the ability to infect humans to induce one of the forms of AIDS, with the essential properties described above, CNCM
Any retrovirus having a genomic RNA capable of hybridizing under stringent conditions with the HIV-2 virus strain (or a cDNA or cDNA fragment derived from that genomic RNA) deposited at It will be understood.

The invention further relates to the respective antigens obtained from HIV-2, in particular purified proteins and glycoproteins. The term “purified” protein or glycoprotein means, respectively, a protein or glycoprotein which produces a single band on polyacrylamide gel electrophoresis, especially under the experimental conditions described above. Any suitable separation and / or purification method may be used to obtain each antigen. An example of a method that can be used is RC MONTELARO et al., J. of Viro-logy June 1982, p.
There is a method described in p1029-1038.

The present invention is generally derived from HIV-2 and
All antigens, in particular proteins, glycoproteins or polypeptides, having immunological properties equivalent to those of the antigenic compounds of. In this case, the two antigens are the same antibody, especially HIV-
2 When recognized by antibodies isolated from the sera of infected patients, or when the two antigens are “immunologically equivalent” as described below.
Two antigens are considered to be "equivalent" when the conditions are met.

A fragment of an antigen (or peptide reconstructed by chemical synthesis) may be considered to be an equivalent polypeptide, protein or glycoprotein so long as it undergoes an immune cross-reactivity with the antigen from which the fragment was sourced. . In other words, the invention includes any polypeptide sharing the same or similar epitope with said antigen that can be recognized by the same antibody. A polypeptide belonging to this type is the expression product of the corresponding sequence of DNA encoding the corresponding polypeptide sequence.

It has been found that the HIV-2 virus can be used as an antigen source for detecting antibodies in all humans contacted with the HIV-2 virus.

The present invention is generally directed to biofluid serum, in particular
It relates to a composition that can be used to diagnose the presence of antibodies against at least one of the antigens of HIV-2 in the serum of humans contacted with HIV-2. This composition is used in place of HIV-1 in the diagnostic method as described in the aforementioned European patent application.
The two extracts, lysates or purified antigens can be used to selectively diagnose the corresponding various AIDS. In this context, the present invention particularly relates to at least one of the internal proteins p12, p16, p26 or p36 or gp140.
According to the composition containing. Examples of these are compositions that simultaneously contain the following proteins:

-P26 and gp36-p26, p36 and gp140-p12, p16 and p26-p16, p26 and gp140 and the like.

It will be clear that these compositions are just a few examples. In particular, the invention relates to viral extracts or lysates containing this group of proteins and / or glycoproteins or all fractions used for the separation of one or more of said proteins or glycoproteins.

The present invention also relates to HIV-2 proteins and / or
Or, a composition comprising a combination of a glycoprotein and a protein and / or glycoprotein of, for example, HIV-1. This combination is, for example, -HUV-1 or HIV-2 core protein, in particular HIV-1 p2
5 and HIV-2 p26, or HIV-1 p18 and HIV-2 p16, -HIV-1 envelope glycoprotein and HIV-2 envelope glycoprotein, especially HIV-1 gp110 and HIV-2. Gp
140, or p42 of HIV-1 and p36 or p42 to p of HIV-2
45 may be used-HIV-1 protein and / or glycoprotein and HIV-2
It may be a mixture with the protein and / or glycoprotein.

Such a diagnostic composition provides a diagnostic means for AIDS or AIDS-related syndromes in which a causative agent covers a wide range. Needless to say, when a composition containing only HIV-2 protein and / or glycoprotein is used as a diagnostic tool, the type of pathogenic retrovirus can be more selectively diagnosed.

The present invention also relates to DNA or DNA fragments, more particularly cloned DNA derived from RNA of HIV-2 retrovirus and DNA fragments obtained from cDNA. The present invention especially relates to sequence homology with all equivalent DNA, in particular with HIV-2 DNA, in particular with at least 50 sequence homology with the sequences coding for the env and pol regions of the HIV-2 strain deposited at the CNCM. %,Preferably
It includes DNA with a range of up to 70%, more preferably up to 90%. In general, the present invention relates to HIV-2 DNA by the "spot blot" method under non-stringent conditions as described above.
Or any equivalent DNA (or
RNA).

The present invention also relates to sera that can be inoculated with HIV-2 to produce in the animal body. Thus, more particularly, the invention relates to polyclonal antibodies specific for each viral antigen, in particular viral proteins or glycoproteins. It also relates to monoclonal antibodies that can be produced by conventional methods and are each more specific for various proteins of HIV-2.

These polyclonal or monoclonal antibodies can be used for various purposes. Its main uses include neutralization of the corresponding protein, inhibition of infectivity of whole virus, and the like. It may also be used, for example, for the detection of viral antigens in biologicals, or for purification of the corresponding proteins and / or glycoproteins, for example on affinity chromatography columns.

In general, the technical literature available on the viruses designated HIV-1 and HTLV-III (in particular the literature referred to in the text) refers to the techniques described in these publications as HIV-2 viruses or equivalent viruses. Included herein as long as they can be used under similar conditions for the isolation of, and can be used under similar conditions to produce various components (particularly proteins, glycoproteins, polypeptides and nucleic acids) from these viruses. Shall be provided. Also, the use of these various ingredients, especially
The teachings of these technical references may also be used for the use of LAS or a corresponding form of AIDS as a diagnostic tool.

The invention particularly relates to a method for the in vitro diagnosis of AIDS. The method involves contacting a serum or other biological medium of a subject to be diagnosed with a composition containing at least one of HIV-2 protein or glycoprotein, an extract of the virus or a lysate, and an immunoreaction. To detect. Examples of such compositions have been given above.

Preferred methods include, for example, ELISA or immunofluorescence type immunoenzymatic reactions. Titers can be measured by direct or indirect immunofluorescence or direct or indirect immunoenzyme assays.

Accordingly, the present invention also includes a viral extract (an extract of one or more HIV-2 viruses alone or one or more
A mixture of HIV-2 virus extract and one or more HIV-1 virus extract). These extracts are labeled. Appropriate labels such as enzymes, fluorescence, radioactivity etc. can be used.

Such titer determination includes, for example, the following processing.

-A specific amount of the extract of the invention or the composition is attached to the wells of a microtiter plate.

-Introducing increasing dilutions of serum into the wells, which mainly contains the antibody whose presence is to be detected in vitro.

-Incubate the microtiter plate.

-Carefully wash the microtiter plate with the appropriate buffer.

-Introduce a labeled antibody specific for human immunoglobulin into the wells of a microtiter plate. The substrate is labeled with a hydrolyzable selective enzyme so that the radiation absorption of the substrate changes at least in a specific wavelength band.

The degree of substrate hydrolysis is preferably detected as a measure of disease latency or pathogenesis by comparison with controls.

The present invention also relates to said diagnostic assembly or kit.

The kit comprises: an extract or a more highly purified fraction of a virus of said type, eg radiolabeled, enzyme labeled or immunofluorescently labeled; an anti-human immunoglobulin or protein A (preferably agarose). Bound to a water-insoluble carrier such as beads),-a lymphocyte extract obtained from a healthy human, -a buffer and optionally a substrate for visualizing a labeling substance.

From the above description, it will be understood that the present invention includes a method for diagnosing HIV-2 virus or its mutant strain, which carries out the various steps described below using the above-mentioned probe. The steps are performed in a specific order such that the characteristic properties of the HIV-2 virus are exerted.

The present invention naturally contemplates the use of HI in serum or another biological fluid or tissue sample of a suspected carrier of HIV-2 virus.
The present invention relates to the use of cDNA or a fragment thereof (or a recombinant containing them) as a probe for diagnosing the presence or absence of V-2 virus. These probes are also preferably labeled (with radioactive, enzyme, fluorescent labels, etc.). A particularly advantageous characteristic of the probe used for carrying out the diagnostic method of HIV-2 virus or mutant strain of HIV-2 is that it contains all or part of cDNA complementary to the genome of HIV-2 virus,
Or, in particular, to include the fragments present in the various clones identified above. In particular, the cDNA of HIV-2 present in clone E2, more particularly the clone
CDNAs containing sequences at the 3'end (LTR) and / or 5'end of the HIV sequence of E2, or the env region of the HIV-2 viral cDNA are preferred.

The probe used in the HIV-2 virus diagnostic method and diagnostic kit of the present invention is not limited to the above-mentioned probe. Conversely, HIV in human biofluids, which is a nucleotide sequence derived from the genome of HIV-2 virus or a mutant strain of HIV-2 or a structurally related virus, is capable of generating one of the forms of AIDS. -2 Includes all nucleotide sequences that allow the antibody to be detected. However,
Of course, the use of nucleotide sequences derived from HIV-2, which is initially human infectious, is preferred.

The detection may be performed by any known method. In particular, serum or other biological media such as cerebrospinal fluid,
A probe is brought into contact with a nucleic acid obtained from cells present in saliva or the like, or the medium itself containing the nucleic acid in a state capable of hybridizing with the probe is brought into contact with the probe. This contact is made under conditions where hybridization of these probes with the nucleic acid can occur.
The resulting hybridization is then detected. If the identification of the type of HIV virus is not necessary, the diagnosis including the hybridization reaction may be carried out using a mixture of probes obtained from HIV-1 or HIV-2, respectively.

In general, a method of diagnosing the presence or absence of HIV-2 virus or a variant thereof in a serum or other fluid or tissue sample of a suspected carrier of HIV-2 virus comprises the following steps.

(1) Prepare a labeled probe.

(2) The DNA of cells in the sample of the suspected carrier is contacted with the labeled probe on a suitable membrane, and hybridization is performed at least once under stringent conditions.

(3) Wash the membrane with a solution that maintains stringency conditions for hybridization.

(4) The presence or absence of HIV-2 virus is detected by an immunodetection method.

According to another preferred embodiment of the method of the present invention, the above-mentioned hybridization is carried out under non-stringent conditions, and the membrane is washed under conditions adapted to the hybridization conditions.

The present invention especially relates to the HIV-2 virus, characterized in that it has a viral RNA corresponding to a cDNA carrying the GAG and ENV genes respectively containing the following nucleotide sequences:

These sequences were obtained by sequencing the corresponding regions of the cDNA corresponding to the genomic HIV-2 ROD. These sequences match the amino acids they encode.

[0239] As mentioned above, the present invention provides that the RNA has similar properties,
In particular, for all HIV-2 viruses having a GAG and ENV region containing a sequence having a nucleotide sequence homology of 50% or more, preferably 70% or more, and more preferably 90% or more with the corresponding GAG and ENV sequences of HIV-2 ROD. Pertain.

[0240] More specifically, the present invention provides that the structure is GAGR
It relates to cDNA fragments encoding p16, p26 and p12, respectively, which are also contained in ODN. The present invention is particularly directed to nucleotides 1 (encoding p16) to nucleotides
405-the sequence of nucleotides 406 to 1155 (encoding p26) -the sequence of nucleotides 1156 to 1566 (encoding p12).

The invention also particularly relates to a cDNA fragment encapsulating ENVR and encoding gp140 from nucleotide 1 to nucleotide 2574.

The present invention further relates to nucleotide sequences which differ from said nucleotide sequences by nucleotide substitutions which utilize the degeneracy of the genetic code such that they do not include modifications of the amino acid sequence encoded by said nucleotide sequences.

Similarly, the present invention provides a protein or glycoprotein having an amino acid sequence corresponding to the above-mentioned amino acid sequence, and an equivalent peptide, that is, addition, substitution or substitution of amino acids that do not affect the overall immunological properties of the peptide. It concerns peptides obtained from the above peptides by deletion.

The invention particularly relates to envelope glycoproteins which exhibit the amino acid sequence encoded by ENVRN.

The present invention also provides 10-500 per kg body weight,
An envelope antigen of HIV-2 virus, preferably HIV-, especially formulated so that a dosage unit of 50-100 mg can be administered.
2 It relates to an immunogenic composition, which comprises gp140 of 2 viruses.

Finally, the present invention provides the protein (p12, p1
6 or p26) or a protein having a structure of gp140 or a predetermined portion of the protein. In the method, a cell host capable of expressing the insert in the vector is appropriately selected, a nucleic acid sequence corresponding to the vector capable of transforming the cell host is inserted, and the selected host is selected by the vector containing the nucleic acid. The cell host transformed and transformed with the modified vector is cultured, and the expressed protein is recovered and purified.

European Patent Application No. 85 905513.9 filed October 18, 1985 for producing a peptide or protein consisting of the expression product of a nucleic acid sequence derived from the genome of HIV-1.
It is also possible to apply the method disclosed in No. 1 to the production of said peptide or protein derived from HIV-2. The description of said European patent application shall be included in the present description as a related art. HIV-2 protein molecular weight
(MP) is shown below in comparison with the molecular weight of HIV-1.

MP (kd) of HIV-2 protein MP (kd) of HIV-1 protein Complete gag 58.3 Complete gag 55.8 p16 15 p16 14.9 p26 27.6 p12 15.8 env 98.6 env 97.4 External env 57.4 Transmembrane env 41.2 HIV- 2 Mir and HIV-2 ROD were released on 9 January 1987 by Salisb
ury (UK) 「National Collection of Animal Cell
Cultures '' (ECACC) with accession number 87 011001 and
It was deposited at 87 011002.

Furthermore, the plasmids pROD35 and pROD27.5
Aberdeen, UK, January 9, 1987, "National Colle
ction of Industrial Bacteria ”(NCIB) with deposit numbers 12398 and 12399, respectively.

All references mentioned herein are intended to be included in the present invention.

1-F. Barre-Sinoussi et al., Science
220, 868 (1983). 2-L. Montagnier et al., In: Human Tcell leukemi
a orlymphoma viruses (Gallo, RC, Essex, ME, Gro
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allo, Science 224, 497 (1984). 4-J. Levy et al., Science 225, 840 (1984). 5-P. Sonigo et al., Cell 42, 369 (1985). 7-JW Curran et al. ., Science 229, 1352 (1985). 8-A. Ellrodt et al., Lancet i, 1383 (1984). 9-P. Piot et al., Lancet ii, 65 (1984). 10-F. Brun- Vezinet et al., Science 226, 453 (198
4). 11−N. Clumeck et al., N. Engl. J. Med. 313, 182.
(1985). 12−AB Rabson and MA Martin, Cell 40, 477 (1
985). 13−S. Benn et al., Science 230, 949 (1985). 14−M. Alizon, Manuscript in preparation. 15−F. Brun-Bezinet, Unpublished data. 16−MD Daniel et al., Science 228, 1201 (1985). 17-PJ Kanki et al., Science 228, 1199 (1985). 18-NL Letwin et al., Science 230, 71 (1985). 19-A. Gatzar et al., Blood 55 , 409 (1980). 20-D. Klatzmann et al., Science 225, 59 (1984). 21-L. Montagier et al., Virology 144, 283 (1985). 22-JS Allan et al., Science 228. , 1091 (1985). 23-F. CHIVel, Manuscript in preparation. 24-F. DiMarzo Veronese et al., Science 229, 1402.
(1985) .25-VS Kalyanarman et al., Science 218, 571 (19
82) .26−ISY Chen, J. McLaughlin, JC Gasson, S.
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[Brief description of drawings]

1A, 1B and 1C are cross-precipitation experiments between sera of patients infected with HIV-1 and HIV-2 and sera of STLV-III infected rhesus monkeys with HIV-1 virus extract. .

FIGS. 2A and 2B show the comparison results of electrophoretic mobilities of respective proteins of HIV-1, HIV-2 and STLV-III in SDS polyacrylamide gel.

FIG. 3 shows the results of cross-hybridization between probes containing the HIV-1, HIV-2 and STLV-III genomic sequences and various subgenomic sequences of the HIV-1 virus.

FIG. 4 is a cDNA derived from HIV-2 ROD RNA.
Shows a restriction map of.

FIG. 5 shows a restriction map of the E2 fragment of cDNA derived from HIV-2. This fragment is HIV-2
Including the region corresponding to the 3'LTR region of.

FIG. 6 shows the nucleotide sequence of a portion of E2. This sequence corresponds to the U3 / R region of HIV-2.

Figure 7: -On the other hand, the structural elements of HIV-1 (Figure 7A) and HIV-1 3'L.
3 shows the sequences derived from the E2 region of the HIV-2 cDNA aligned with the region containing the TR, -on the other hand in view of the sequences derived from the E2 region of HIV-2 and the large number of deletions and insertions, HI aligned to the array
The common nucleotide present in the corresponding sequence of V-1 is shown (Fig. 7B).

FIG. 8 schematically shows the structures of several clones of phage (clones ROD4, ROD27 and ROD35) modified by some insertions derived from HIV-2 derived cDNA. The sequences from ROD4, ROD27 and ROD35 subcloned in plasmid pUC18 are also shown schematically in the figure. Subcloning sequences are original ROD4, RO
It is arranged so as to correspond to the areas of D27 and ROD35.

FIG. 9: (a) Present in 11 fragments taken from different regions of the complete HIV-1 genome (the fragments are shown schematically at the bottom of the figure) and ROD4. Shows the relative intensities of hybridization between the HIV-2 cDNA and (b) a fragment obtained from HIV-2 with the same cDNA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 5/24 7/02 8931-4B 7/04 8931-4B 15/49 A C12P 21/02 C 8214-4B C12Q 1/70 7823-4B G01N 33/50 P 7055-2J 33/53 D 8310-2J 33/569 H 9015-2J 33/577 B 9015-2J // (C12P 21/02 C12R 1:91 ) (31) Priority claim number 86 01985 (32) Priority date February 13, 1986 (33) Priority claiming country France (FR) (31) Priority claim number 86 038881 (32) Priority date March 1986 18th (33) Priority claim France (FR) (31) Priority claim number 86 04215 (32) Priority date March 24, 1986 (33) Priority claim France (FR) (31) Priority claim No. 835228 (32) Priority date March 3, 1986 Date (33) Priority claiming United States (US) (31) Priority claiming number 91080 (32) Priority date October 6, 1986 (33) Priority claiming United States (US) (31) Priority claiming number 933184 (32) Priority date November 21, 1986 (33) Priority claiming country United States (US) (72) Inventor Donnie's Getar France, 75015 Paris, Liu Anselm Payyan, 4 Be (72) Inventor Marc Arison France, 75005 Paris, Lieuil de You Cardinal Lemoine, 71 (72) Inventor François Clavelle France, 75016 Paris, Riu de La Sonpecion, 83 (72) Inventor Mireille Guille-Hyader France, 75016 Paris, Squall Rocamadoeur, 2 (72) Inventor Pierre Sonigo France, 75015 Paris, Lieu Guillebert, 23 (72) Inventor Françoise Blanc-Vezine France, 75 005Paris, Boulevard Saint-Giermain, 24 (72) Inventor Marianne Rei France, 75004 Paris, Riuyu Dou Tample, 10 (72) Inventor Christine Rigiu France, 75015 Paris, Riuille Dou Dansig, 21 (72) Inventor Christine Katrama, France, 75004 Paris, Rouille du Giarant, 8

Claims (25)

[Claims] 1. Essential morphology and immunology of a retrovirus that is infectious to human T4 lymphocytes and has been deposited with the CNCM under accession numbers I-502, I-532, I-642 or I-643. HIV-2 retrovirus or its mutant having specific characteristics. 2. Having the following characteristics, namely: -targeting human Leu3 cells (or T4 lymphocytes) or permanent cell lines derived from said T4 lymphocytes as suitable targets; -infected human T4 lymphocytes. Cytotoxicity against polyadenylate oligodeoxythymidylate (polyadenylate
(A) -oligo (dT) 12-18) with a strong affinity for Mg ²⁺
Have reverse transcriptase activity that requires the presence of ions, -a density in the sucrose gradient of about 1.16, -an average diameter of 140 nm, an average core diameter of 41 nm, -express T4 protein Being able to culture in a permanent cell line, -not infecting T8 lymphocytes, -the lysate is p24 of HTLV-1 or HTLV-2 virus
Comprising a p26 protein which does not immunologically cross-react with the protein, said lysate further comprising a p16 protein which is not immunologically recognized by the HTLV-1 or HTLV-2 p19 protein in a radioimmunoprecipitation assay, -The lysate further contains an envelope glycoprotein having a molecular weight of about 130,000 to 140,000 which does not immunologically cross-react with gp110 of HTLV-1 retrovirus; -the lysate further has an apparent molecular weight of about 36,000 and ³⁵ S- Includes a molecule that can be labeled with cysteine, -genomic RNA does not hybridize with HIV-1 genomic RNA, ENV gene, or LTR under stringent conditions, -genomic RNA under non-stringent conditions HIV-
1. The purified retrovirus according to claim 1, which hybridizes weakly with the nucleotide sequence of the GAG region of one genome. 3. The lysate further has an apparent molecular weight of 42,000 to 4
Claim 2 characterized in that it comprises a molecule with 5,000
Retrovirus according to. 4. The nucleotide sequence of genomic RNA containing an R region and a U3 region further comprises a nucleotide sequence corresponding to the following nucleotide sequence:
The retrovirus according to any one of 1 to 3. GTGGAAGGCGAGACTGAAAGCAAGAGGAATACCATTTAGTTAAAGGACAG GAACAGCTATACTTGGTCAGGGCAGGAAGTAACTAACAGAAACAGCTGAG ACTGCAGGGACTTTCCAGAAGGGGCTGTAACCAAGGGAGGGACATGGGAG GAGCTGGTGGGGAACGCCTCATATTCTCTGTATAATATACCCGCTGCTTG CATTGTACTTCAGTCGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTGGAG GATCTCTCCAGCACTAGACGGATGAGCCTGGGTGCCCTGCTAGACTCTCA CCAGCACTTGGCCGGTGCTGGCAGACGGCCCCACGCTTGCCTGCTTAAAA ACCTTCCTTAATAAAGCTGCAGTAGAAGCA 5. The retrovirus according to any one of claims 1 to 4, wherein the genomic RNA further contains a GAG sequence corresponding to the following nucleotide sequences. 6. The retrovirus according to any one of claims 1 to 5, wherein the genomic RNA contains an ENV sequence corresponding to the following nucleotide sequences. 7. The nucleotide sequence of HIV-1 ENV gene and LTR close to the gene, particularly HIV-1 nucleotide sequence 5290-913.
0 and the sequence of the POL region of the HIV-1 genome, especially HIV-1
7. The retrovirus according to any one of claims 1 to 6, which does not substantially hybridize with any of the nucleotide sequences 2170-2240 of. 8. An HIV-2 retrovirus envelope glycoprotein, such as gp140, or a portion of said glycoprotein, together with a pharmaceutically acceptable vehicle suitable as a component of an HIV-2 effective vaccine. An immunogenic composition comprising: 9. The composition of claim 8, comprising at least a portion of an immunogenic glycoprotein comprising a protein scaffold of the sequence: ENVRN MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAlaSerAlaCys LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThrArgAsn ArgAspThrTrpGlyThrIleGlnCysLeuProAspAsnAspAsp TyrGlnGluIleThrLeuAsnValThrGluAlaPheAspAlaTrp AsnAsnThrValThrGluGlnAlaIleGluAspValTrpHisLeu PheGluThrSerIleLysProCysValLysLeuThrProLeuCys ValAlaMetLysCysSerSerThrGluSerSerThrGlyAsnAsn ThrThrSerLysSerThrSerThrThrThrThrThrProThrAsp GlnGluGlnGluIleSerGluAspThrProCysAlaArgAlaAsp AsnCysSerGlyLeuGlyGluGluGluThrIleAsnCysGlnPhe AsnMetThrGlyLeuGluArgAspLysLysLysGlnTyrAsnGlu ThrTrpTyrSerLysAspValValCysGluThrAsnAsnSerThr AsnGlnThrGlnCysTyrMetAsnHisCysAsnThrSerValIle ThrGluSerCysAspLysHisTyrTrpAspAlaIleArgPheArg TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr AsnTyrSerGlyPheAlaProAsnCysSerLysValValAlaSer ThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGly PheAsnGlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHis GlyArgAspAsnArgThrIleIleSerLeuAsnLysTyrTyrAsn LeuSerLeuHisCysLysArgProGlyAsnLysThrValLysGln IleMetLeuMetSerGlyHisValPheH isSerHisTyrGlnPro IleAsnLysArgProArgGlnAlaTrpCysTrpPheLysGlyLys TrpLysAspAlaMetGlnGluValLysThrLeuAlaLysHisPro ArgTyrArgGlyThrAsnAspThrArgAsnIleSerPheAlaAla ProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsn CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn TrpIleGluAsnLysThrHisArgAsnTyrAlaProCysHisIle LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr SerIleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsn IleThrPheSerAlaGluValAlaGluLeuTyrArgLeuGluLeu GlyAspTyrLysLeuValGluIleThrProIleGlyPheAlaPro ThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArg GlyValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGly SerAlaMetGlyAlaArgAlaSerLeuThrValSerAlaGlnSer ArgThrLeuLeuAlaGlyIleValGlnGlnGlnGlnGlnLeuLeu AspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrp GlyThrLysAsnLeuGLnAlaArgValThrAlaIleGluLysTyr LeuGlnAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArg GlnValCysHisThrThrValProTrpValAsnAspSerLeuAla ProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGlnVal ArgTyrLeuGluAlaAsnIleSerLysSerLeuGluGlnAlaGln IleGlnGlnGluLysA snMetTyrGluLeuGlnLysLeuAsnSer TrpAspIlePheGlyAsnTrpPheAspLeuThrSerTrpValLys TyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeu ArgIleValIleTyrValValGlnMetLeuSerArgLeuArgLys GlyTyrArgProValPheSerSerProProGlyTyrIleGlnGln IleHisIleHisLysAspArgGlyGlnProAlaAsnGluGluThr GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrp ProIleAlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeu LeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSer PheLeuThrLeuGlnLeuIleTyrGlnAsnLeuArgAspTrpLeu ArgLeuArgThrAlaPheLeuGlnTyrGlyCysGluTrpIleGln GluAlaPheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAla GlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArg GlyIleLeuAlaValProArgArgIleArgGlnGlyAlaGluIle AlaLeuLeu *** GlyThrAlaValSerAlaGlyArgLeuTyrGlu TyrSerMetGluGlyProSerSerArgLysGlyGluLysPheVal GlnAlaThrLysTyrGly 10. A body weight of 10 to 500, in particular 50 to 10 per kg.
The immunogenic composition according to claim 8 or 9, wherein the antigen is mixed so that 0 µg of the dosage unit can be administered. 11. An antigen comprising the following amino acid sequence recognized by an anti-p12 antibody or a part of the sequence: Anti p16 antibody antigen comprising a portion of the following amino acid sequence or sequence that is recognized by MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGlu LeuGluArgIleArgLeuArgProGlyGlyLysLysLysTyrArg LeuLysHisIleValTrpAlaAlaAsnLysLeuAspArgPheGly LeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLysIle LeuThrValLeuAspProMetValProThrGlySerGluAsnLeu LysSerLeuPheAsnThrValCysValIleTrpCysIleHisAla GluGluLysValLysAspThrGluGlyAlaLysGlnIleValArg ArgHisLeuValAlaGluThrGlyThrAlaGluLysMetProSer ThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr, antigen comprising a portion of the following amino acid sequence or sequence that is recognized by anti-p26 antibody ProValGlnHisValGlyGlyAsnTyrThrHisIleProLeuSer ProArgThrLeuAsnAlaTrpValLysLeuValGluGluLysLys PheGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly CysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAsp HisGlnAlaAlaMetGlnIleIleArgGluIleIleAsnGluGlu AlaAlaGluTrpAspValGlnHisProIleProGlyProLeuPro AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr ThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGln AsnProValProValGlyAsnIleTyrArgArgTrpIleGlnIle GlyLeuGlnLysCysValArgMetTyrAsnProThrAsnIleLeu AspIleLysGlnGlyProLysGluProPheGlnSerTyrValAsp ArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaVal LysAsnTrpMetThrGlnThrLeuLeuValGlnAsnAlaAsnPro AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGln LysAlaArgLeuMetAlaGluAlaLeuLysGluValIleGlyPro AlaProIleProPheAlaAlaAlaGlnGln and antigen comprises a portion of the following amino acid sequence or sequence that is recognized by anti-gp140 antibodies ENVRN MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAlaSerAlaCys LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThrArgAsn ArgAspThrTrpGlyThrIleGlnCysLeuProAspAsnAspAsp TyrGlnGluIleThrLeuAsnValThrGluAlaPheAspAlaTrp AsnAsnThrValThrGluGlnAlaIleGluAspValTrpHisLeu PheGluThrSerIleLysProCysValLysLeuThrProLeuCys ValAlaMetLysCysSerSerThrGluSerSerThrGlyAsnAsn ThrThrSerLysSerThrSerThrThrThrThrThrProThrAsp GlnGluGlnGluIleSerGluAspThrProCysAlaArgAlaAsp AsnCysSerGlyLe uGlyGluGluGluThrIleAsnCysGlnPhe AsnMetThrGlyLeuGluArgAspLysLysLysGlnTyrAsnGlu ThrTrpTyrSerLysAspValValCysGluThrAsnAsnSerThr AsnGlnThrGlnCysTyrMetAsnHisCysAsnThrSerValIle ThrGluSerCysAspLysHisTyrTrpAspAlaIleArgPheArg TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr AsnTyrSerGlyPheAlaProAsnCysSerLysValValAlaSer ThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGly PheAsnGlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHis GlyArgAspAsnArgThrIleIleSerLeuAsnLysTyrTyrAsn LeuSerLeuHisCysLysArgProGlyAsnLysThrValLysGln IleMetLeuMetSerGlyHisValPheHisSerHisTyrGlnPro IleAsnLysArgProArgGlnAlaTrpCysTrpPheLysGlyLys TrpLysAspAlaMetGlnGluValLysThrLeuAlaLysHisPro ArgTyrArgGlyThrAsnAspThrArgAsnIleSerPheAlaAla ProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsn CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn TrpIleGluAsnLysThrHisArgAsnTyrAlaProCysHisIle LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr SerIleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsn IleThrPheSerAlaGluValAlaGluLeuTyrArgLeuGluLeu Gl yAspTyrLysLeuValGluIleThrProIleGlyPheAlaPro ThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArg GlyValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGly SerAlaMetGlyAlaArgAlaSerLeuThrValSerAlaGlnSer ArgThrLeuLeuAlaGlyIleValGlnGlnGlnGlnGlnLeuLeu AspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrp GlyThrLysAsnLeuGLnAlaArgValThrAlaIleGluLysTyr LeuGlnAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArg GlnValCysHisThrThrValProTrpValAsnAspSerLeuAla ProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGlnVal ArgTyrLeuGluAlaAsnIleSerLysSerLeuGluGlnAlaGln IleGlnGlnGluLysAsnMetTyrGluLeuGlnLysLeuAsnSer TrpAspIlePheGlyAsnTrpPheAspLeuThrSerTrpValLys TyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeu ArgIleValIleTyrValValGlnMetLeuSerArgLeuArgLys GlyTyrArgProValPheSerSerProProGlyTyrIleGlnGln IleHisIleHisLysAspArgGlyGlnProAlaAsnGluGluThr GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrp ProIleAlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeu LeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSer PheLeuThrLeuGlnLeuIleTyrGlnAsnLeuArgAspTrpLeu ArgLeuArgThrAlaPheLeuGlnTyrGlyCysGlu TrpIleGln GluAlaPheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAla GlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArg GlyIleLeuAlaValProArgArgIleArgGlnGlyAlaGluIle AlaLeuLeu *** GlyThrAlaValSerAlaGlyArgLeuTyrGlu TyrSerMetGluGlyProSerSerArgLysGlyGluLysPheVal GlnAlaThrLysTyrGly either monoclonal antibody characterized by having the ability to specifically recognize one of the antigens. 12. A hybridoma that secretes the monoclonal antibody according to claim 11. 13. A nucleic acid which is derived from at least a part of RNA of one of the HIV-2 virus or its mutant and is optionally labeled. 14. The complete genome RN of HIV-2 retrovirus
The nucleic acid according to claim 13, comprising at least a part of a cDNA corresponding to A. 15. The nucleic acid according to claim 13, which comprises the following nucleotide sequence: GTGGAAGGCGAGACTGAAAGCAAGAGGAATACCATTTAGTTAAAGGACAG GAACAGCTATACTTGGTCAGGGCAGGAAGTAACTAACAGAAACAGCTGAG ACTGCAGGGACTTTCCAGAAGGGGCTGTAACCAAGGGAGGGACATGGGAG GAGCTGGTGGGGAACGCCTCATATTCTCTGTATAATATACCCGCTGCTTG CATTGTACTTCAGTCGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTGGAG GATCTCTCCAGCACTAGACGGATGAGCCTGGGTGCCCTGCTAGACTCTCA CCAGCACTTGGCCGGTGCTGGCAGACGGCCCCACGCTTGCCTGCTTAAAA ACCTTCCTTAATAAAGCTGCAGTAGAAGCA 16. The nucleic acid according to claim 13, comprising a nucleotide sequence encoding at least a part of the following amino acid sequence. GAGRODN MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGLu LeuGluArgIleArgLeuArgProGlyGlyLysLysLysTyrArg LeuLysHisIleValTrpAlaAlaAsnLysLeuAspArgPheGly LeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLysIle LeuThrValLeuAspProMetValProThrGlySerGluAsnLeu LysSerLeuPheAsnThrValCysValIleTrpCysIleHisAla GluGluLysValLysAspThrGluGlyAlaLysGlnIleValArg ArgHisLeuValAlaGluThrGlyThrAlaGluLysMetProSer ThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr ProValGlnHisValGlyGlyAsnTyrThrHisIleProLeuSer ProArgThrLeuAsnAlaTrpValLysLeuValGluGluLysLys PheGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly CysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAsp HisGlnAlaAlaMetGlnIleIleArgGluIleIleAsnGluGlu AlaAlaGluTrpAspValGlnHisProIleProGlyProLeuPro AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr ThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGln AsnProValProValGlyAsnIleTyrArgArgTrpIleGlnIle GlyLeuGlnLysCysValArgMetTyrAsnProThrAsnIleLeu AspIleLysGlnGlyProLysGluProPheGlnSerTyrValAsp ArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaVal LysAsnTrpMetThrGlnThrLeuLe uValGlnAsnAlaAsnPro AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGln LysAlaArgLeuMetAlaGluAlaLeuLysGluValIleGlyPro AlaProIleProPheAlaAlaAlaGlnGlnArgLysAlaPheLys CysTrpAsnCysGlyLysGluGlyHisSerAlaArgGlnCysArg AlaProArgArgGlnGlyCysTrpLysCysGlyLysProGlyHis IleMetThrAsnCysProAspArgGlnAlaGlyPheLeuGlyLeu GlyProTrpGlyLysLysProArgAsnPheProValAlaGlnVal ProGlnGlyLeuThrProThrAlaProProValAspProAlaVal AspLeuLeuGluLysTyrMetGlnGlnGlyLysArgGlnArgGlu GlnArgGluArgProTyrLysGluValThrGluAspLeuLeuHis LeuGluGlnGlyGluThrProTyrArgGluProProThrGluAsp LeuLeuHisLeuAsnSerLeuPheGlyLysAspGln 17. The nucleic acid according to claim 13, which comprises a nucleotide sequence encoding at least a part of the following amino acid sequence. 18. The nucleic acid according to claim 13, which comprises a nucleotide sequence encoding at least a part of the following amino acid sequence. MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGlu LeuGluArgIleArgLeuArgProGlyGlyLysLysLysTyrArg LeuLysHisIleValTrpAlaAlaAsnLysLeuAspArgPheGly LeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLysIle LeuThrValLeuAspProMetValProThrGlySerGluAsnLeu LysSerLeuPheAsnThrValCysValIleTrpCysIleHisAla GluGluLysValLysAspThrGluGlyAlaLysGlnIleValArg ArgHisLeuValAlaGluThrGlyThrAlaGluLysMetProSer ThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr 19. The nucleic acid according to claim 13, which comprises a nucleotide sequence encoding at least a part of the following amino acid sequence. ProValGlnHisValGlyGlyAsnTyrThrHisIleProLeuSer ProArgThrLeuAsnAlaTrpValLysLeuValGluGluLysLys PheGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly CysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAsp HisGlnAlaAlaMetGlnIleIleArgGluIleIleAsnGluGlu AlaAlaGluTrpAspValGlnHisProIleProGlyProLeuPro AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr ThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGln AsnProValProValGlyAsnIleTyrArgArgTrpIleGlnIle GlyLeuGlnLysCysValArgMetTyrAsnProThrAsnIleLeu AspIleLysGlnGlyProLysGluProPheGlnSerTyrValAsp ArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaVal LysAsnTrpMetThrGlnThrLeuLeuValGlnAsnAlaAsnPro AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGln LysAlaArgLeuMetAlaGluAlaLeuLysGluValIleGlyPro AlaProIleProPheAlaAlaAlaGlnGln 20. The nucleic acid according to claim 13, comprising a nucleotide sequence encoding at least a part of the following amino acid sequence. ENVRN MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAlaSerAlaCys LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThrArgAsn ArgAspThrTrpGlyThrIleGlnCysLeuProAspAsnAspAsp TyrGlnGluIleThrLeuAsnValThrGluAlaPheAspAlaTrp AsnAsnThrValThrGluGlnAlaIleGluAspValTrpHisLeu PheGluThrSerIleLysProCysValLysLeuThrProLeuCys ValAlaMetLysCysSerSerThrGluSerSerThrGlyAsnAsn ThrThrSerLysSerThrSerThrThrThrThrThrProThrAsp GlnGluGlnGluIleSerGluAspThrProCysAlaArgAlaAsp AsnCysSerGlyLeuGlyGluGluGluThrIleAsnCysGlnPhe AsnMetThrGlyLeuGluArgAspLysLysLysGlnTyrAsnGlu ThrTrpTyrSerLysAspValValCysGluThrAsnAsnSerThr AsnGlnThrGlnCysTyrMetAsnHisCysAsnThrSerValIle ThrGluSerCysAspLysHisTyrTrpAspAlaIleArgPheArg TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr AsnTyrSerGlyPheAlaProAsnCysSerLysValValAlaSer ThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGly PheAsnGlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHis GlyArgAspAsnArgThrIleIleSerLeuAsnLysTyrTyrAsn LeuSerLeuHisCysLysArgProGlyAsnLysThrValLysGln IleMetLeuMetSerGlyHisValPheH isSerHisTyrGlnPro IleAsnLysArgProArgGlnAlaTrpCysTrpPheLysGlyLys TrpLysAspAlaMetGlnGluValLysThrLeuAlaLysHisPro ArgTyrArgGlyThrAsnAspThrArgAsnIleSerPheAlaAla ProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsn CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn TrpIleGluAsnLysThrHisArgAsnTyrAlaProCysHisIle LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr SerIleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsn IleThrPheSerAlaGluValAlaGluLeuTyrArgLeuGluLeu GlyAspTyrLysLeuValGluIleThrProIleGlyPheAlaPro ThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArg GlyValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGly SerAlaMetGlyAlaArgAlaSerLeuThrValSerAlaGlnSer ArgThrLeuLeuAlaGlyIleValGlnGlnGlnGlnGlnLeuLeu AspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrp GlyThrLysAsnLeuGLnAlaArgValThrAlaIleGluLysTyr LeuGlnAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArg GlnValCysHisThrThrValProTrpValAsnAspSerLeuAla ProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGlnVal ArgTyrLeuGluAlaAsnIleSerLysSerLeuGluGlnAlaGln IleGlnGlnGluLysA snMetTyrGluLeuGlnLysLeuAsnSer TrpAspIlePheGlyAsnTrpPheAspLeuThrSerTrpValLys TyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeu ArgIleValIleTyrValValGlnMetLeuSerArgLeuArgLys GlyTyrArgProValPheSerSerProProGlyTyrIleGlnGln IleHisIleHisLysAspArgGlyGlnProAlaAsnGluGluThr GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrp ProIleAlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeu LeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSer PheLeuThrLeuGlnLeuIleTyrGlnAsnLeuArgAspTrpLeu ArgLeuArgThrAlaPheLeuGlnTyrGlyCysGluTrpIleGln GluAlaPheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAla GlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArg GlyIleLeuAlaValProArgArgIleArgGlnGlyAlaGluIle AlaLeuLeu *** GlyThrAlaValSerAlaGlyArgLeuTyrGlu TyrSerMetGluGlyProSerSerArgLysGlyGluLysPheVal GlnAlaThrLysTyrGly 21. The nucleic acid according to any one of claims 14 to 20, which is a recombinant nucleic acid containing a nucleic acid obtained from a vector into which the cDNA or a part of the cDNA is inserted. 22. The recombinant nucleic acid according to claim 21, which is labeled. 23. For the detection of HIV-2 retroviruses or RNAs thereof in biological fluids or tissues, in particular human LAS.
Or in order to diagnose the latency or onset of AIDS in vitro,
A nucleic acid contained in the biological fluid or tissue, and Claim 14.
To the probe containing the nucleic acid of any one of 22 to 22 under stringent hybridization conditions for a time necessary for the hybridization to occur, and the formed hybrid is washed with a solution that ensures the maintenance of the stringent conditions. , Detecting the hybrid formed. 24. Human T4 lymphocytes or T4 derived from said T4 lymphocytes for producing HIV-2 retrovirus.
Permanent cell lines containing the phenotype are cultured after being pre-infected with an isolate of HIV-2 virus, especially in the medium of the lymphocytes or cell lines when the reverse transcriptase activity level reaches a predetermined threshold. The amount of virus released is especially sucrose or metr
A method characterized by collecting and purifying by fractional centrifugation in an izamide gradient. 25. To produce a hybridization probe for detecting RNA of HIV-2 retrovirus,
DNA sequence, especially the DN of any of claims 13 to 21
A method comprising inserting the A sequence into a cloning vector by in vitro recombination, cloning the obtained modified vector in a competent cell host, and recovering the obtained recombinant DNA.